The primary function of DNA replication is understood to be the provision of progeny with the genetic information possessed by the parent. Thus, the replication of DNA must be complete and carried out in such a way as to maintain genetic stability within the organism and the species. The process of DNA replication is complex and involves many cellular functions and several verification procedures to ensure fidelity in replication. About 30 proteins are involved in the replication of the Escherichia coli chromosome, and this process is more complex in eukaryotic organisms. The first enzymologic observations on DNA replication were made by Arthur Kornberg, who described in E coli the existence of a replication enzyme now called DNA polymerase I. This enzyme has multiple catalytic activities, a complex structure, and a requirement for the triphosphates of the four deoxyribonucleosides of adenine, guanine, cytosine, and thymine. The polymerization reaction catalyzed by DNA polymerase I of E coli has served as a prototype for all DNA polymerases of both prokaryotes and eukaryotes, even though it is now recognized that the major role of this polymerase is proofreading and repair.
In all cells, replication can occur only from a single-stranded DNA (ssDNA) template. Therefore, mechanisms must exist to target the site of initiation of replication and to unwind the dsDNA in that region. The replication complex must then form. After replication is complete in an area, the parent and daughter strands must re-form dsDNA. In eukaryotic cells, an additional step must occur. The dsDNA must re-form the chromatin structure, including nucleosomes that existed prior to the onset of replication. Although this entire process is not completely understood in eukaryotic cells, replication has been quite precisely described in prokaryotic cells, and the general principles are the same in both. The major steps are listed in Table 35–4, illustrated in Figure 35–13, and discussed, in sequence, below. A number of proteins, most with specific enzymatic action, are involved in this process (Table 35–5).
TABLE 35–4Steps Involved in DNA Replication in Eukaryotes ||Download (.pdf) TABLE 35–4 Steps Involved in DNA Replication in Eukaryotes
|1. Identification of the origins of replication |
|2. ATP hydrolysis-driven unwinding of dsDNA to provide an ssDNA template |
|3. Formation of the replication fork; synthesis of RNA primer |
|4. Initiation of DNA synthesis and elongation |
|5. Formation of replication bubbles with ligation of the newly synthesized DNA segments |
|6. Reconstitution of chromatin structure |
TABLE 35–5Classes of Proteins Involved in Replication ||Download (.pdf) TABLE 35–5 Classes of Proteins Involved in Replication
|Protein ||Function |
|DNA polymerases ||Deoxynucleotide polymerization |
|Helicases ||ATP-driven processive unwinding of DNA |
|Topoisomerases ||Relieve torsional strain that results from helicase-induced unwinding |
|DNA primase ||Initiates synthesis of RNA primers |
|Single-strand binding proteins (SSBs) ||Prevent premature reannealing of dsDNA |
|DNA ligase ||Seals the single strand nick between the nascent chain and Okazaki fragments on lagging strand |
Steps involved in DNA replication. This figure describes DNA replication in an E coli cell, but the general steps are similar in eukaryotes. A specific interaction of a protein (the dnaA protein) to the origin of replication (oriC) results in local unwinding of DNA at an adjacent A+T-rich region. The DNA in this area is maintained in the single-strand conformation (ssDNA) by single-strand-binding proteins (SSBs). This allows a variety of proteins, including helicase, primase, and DNA polymerase, to bind and to initiate DNA synthesis. The replication fork proceeds as DNA synthesis occurs continuously (long red arrow) on the leading strand and discontinuously (short black arrows) on the lagging strand. The nascent DNA is always synthesized in the 5′ to 3′ direction, as DNA polymerases can add a nucleotide only to the 3′ end of a DNA strand.
The Origin of Replication
At the origin of replication (ori), there is an association of sequence-specific dsDNA-binding proteins with a series of direct repeat DNA sequences. In bacteriophage λ, the oriλ is bound by the λ-encoded O protein to four adjacent sites. In E coli, the oriC is bound by the protein dnaA. In both cases, a complex is formed consisting of 150 to 250 bp of DNA and multimers of the DNA-binding protein. This leads to the local denaturation and unwinding of an adjacent A+T-rich region of DNA. Functionally similar autonomously replicating sequences (ARS) or replicators have been identified in yeast cells. The ARS contains a somewhat degenerate 11-bp sequence called the origin replication element (ORE). The ORE binds a set of proteins, analogous to the dnaA protein of E coli, the group of proteins is collectively called the origin recognition complex (ORC). ORC homologs have been found in all eukaryotes examined. The ORE is located adjacent to an approximately 80-bp A+T-rich sequence that is easy to unwind. This is called the DNA unwinding element (DUE). The DUE is the origin of replication in yeast and is bound by the MCM protein complex.
Consensus sequences similar to ori or ARS in structure have not been precisely defined in mammalian cells, though several of the proteins that participate in ori recognition and function have been identified and appear quite similar to their yeast counterparts in both amino acid sequence and function.
The interaction of proteins with ori defines the start site of replication and provides a short region of ssDNA essential for initiation of synthesis of the nascent DNA strand. This process requires the formation of a number of protein-protein and protein-DNA interactions. A critical step is provided by a DNA helicase that allows for processive unwinding of DNA. In uninfected E coli, this function is provided by a complex of dnaB helicase and the dnaC protein. Single-stranded DNA-binding proteins (SSBs) stabilize this complex. In λ phage-infected bacterial cells the phage protein P binds to dnaB and the P/dnaB complex binds to oriλ by interacting with the O protein. dnaB is not an active helicase when in the P/dnaB/O complex. Three E coli heat shock proteins (dnaK, dnaJ, and GrpE) cooperate to remove the P protein and activate the dnaB helicase. In cooperation with SSB, this leads to DNA unwinding and active replication. In this way, the replication of the λ phage is accomplished at the expense of replication of the host E coli cell.
Formation of the Replication Fork
A replication fork consists of four components that form in the following sequence: (1) the DNA helicase unwinds a short segment of the parental duplex DNA; (2) a primase initiates synthesis of an RNA molecule that is essential for priming DNA synthesis; (3) the DNA polymerase initiates nascent, daughter-strand synthesis; and (4) SSBs bind to ssDNA and prevent premature reannealing of ssDNA to dsDNA. These reactions are illustrated in Figure 35–13.
The DNA polymerase III enzyme (the dnaE gene product in E coli) binds to template DNA as part of a multiprotein complex that consists of several polymerase accessory factors (β, γ, δ, δ′, and τ). DNA polymerases only synthesize DNA in the 5′ to 3′ direction, and only one of the several different types of polymerases is involved at the replication fork. Because the DNA strands are antiparallel (see Chapter 34), the polymerase functions asymmetrically. On the leading (forward) strand, the DNA is synthesized continuously. On the lagging (retro-grade) strand, the DNA is synthesized in short (1-5 kb; see Figure 35–16) fragments, the so-called Okazaki fragments, so named after the scientist who discovered them. Several Okazaki fragments (up to a thousand) must be sequentially synthesized for each replication fork. To ensure that this happens, the helicase acts on the lagging strand to unwind dsDNA in a 5′ to 3′ direction. The helicase associates with the primase to afford the latter proper access to the template. This allows the RNA primer to be made and, in turn, the polymerase to begin replicating the DNA. This is an important reaction sequence since DNA polymerases cannot initiate DNA synthesis de novo. The mobile complex between helicase and primase has been called a primosome. As the synthesis of an Okazaki fragment is completed and the polymerase is released, a new primer has been synthesized. The same polymerase molecule remains associated with the replication fork and proceeds to synthesize the next Okazaki fragment.
The DNA Polymerase Complex
A number of different DNA polymerase molecules engage in DNA replication. These share three important properties: (1) chain elongation, (2) processivity, and (3) proofreading. Chain elongation accounts for the rate (in nucleotides per second; nt/s) at which polymerization occurs. Processivity is an expression of the number of nucleotides added to the nascent chain before the polymerase disengages from the template. The proofreading function identifies copying errors and corrects them. In E coli, DNA polymerase III (pol III) functions at the replication fork. Of all polymerases, it catalyzes the highest rate of chain elongation and is the most processive. It is capable of polymerizing 0.5 Mb of DNA during one cycle on the leading strand. Pol III is a large (>1 MDa), multisubunit protein complex in E coli. DNA pol III associates with the two identical β subunits of the DNA sliding “clamp”; this association dramatically increases pol III-DNA complex stability, processivity (100 to >50,000 nucleotides) and rate of chain elongation (20-50 nt/s) generating the high degree of processivity the enzyme exhibits.
Polymerase I (pol I) and II (pol II) are mostly involved in proofreading and DNA repair. Eukaryotic cells have counterparts for each of these enzymes plus a large number of additional DNA polymerases primarily involved in DNA repair. A comparison is shown in Table 35–6.
TABLE 35–6A Comparison of Prokaryotic and Eukaryotic DNA Polymerases ||Download (.pdf) TABLE 35–6 A Comparison of Prokaryotic and Eukaryotic DNA Polymerases
|E coli ||Eukaryotic ||Function |
|I || ||Gap filling following DNA replication, repair, and recombination |
|II || ||DNA proofreading and repair |
| ||β ||DNA repair |
| ||γ ||Mitochondrial DNA synthesis |
|III ||ε ||Processive, leading strand synthesis |
|DnaG ||α ||Primase |
| ||δ ||Processive, lagging strand synthesis |
In mammalian cells, the polymerase is capable of polymerizing at a rate that is somewhat slower than the rate of polymerization of deoxynucleotides by the bacterial DNA polymerase complex. This reduced rate may result from interference by nucleosomes.
Initiation & Elongation of DNA Synthesis
The initiation of DNA synthesis (Figure 35–14) requires priming by a short length of RNA, about 10 to 200 nucleotides long. In E coli this is catalyzed by dnaG (primase), in eukaryotes DNA Pol α synthesizes these RNA primers. The priming process involves nucleophilic attack by the 3′-hydroxyl group of the RNA primer on the phosphate of the first entering deoxynucleoside triphosphate (N in Figure 35–14) with the splitting off of pyrophosphate; this transition to DNA synthesis is catalyzed by the appropriate DNA polymerases (DNA pol III in E coli; DNA pol δ and ε in eukaryotes). The 3′-hydroxyl group of the recently attached deoxyribonucleoside monophosphate is then free to carry out a nucleophilic attack on the next entering deoxyribonucleoside triphosphate (N + 1 in Figure 35–14), again at its α phosphate moiety, with the splitting off of pyrophosphate. Of course, selection of the proper deoxyribonucleotide whose terminal 3′-hydroxyl group is to be attacked is dependent upon proper base pairing with the other strand of the DNA molecule according to Watson and Crick base pairing rules (Figure 35–15). When an adenine deoxyribonucleoside monophosphoryl moiety is in the template position, a thymidine triphosphate will enter and its α phosphate will be attacked by the 3′-hydroxyl group of the deoxyribonucleoside monophosphoryl most recently added to the polymer. By this stepwise process, the template dictates which deoxyribonucleoside triphosphate is complementary and by hydrogen bonding holds it in place while the 3′-hydroxyl group of the growing strand attacks and incorporates the new nucleotide into the polymer. These segments of DNA attached to an RNA initiator component are the Okazaki fragments (Figure 35–16). In mammals, after many Okazaki fragments are generated, the replication complex begins to remove the RNA primers, to fill in the gaps left by their removal with the proper base-paired deoxynucleotide, and then to seal the fragments of newly synthesized DNA by enzymes referred to as DNA ligases.
The initiation of DNA synthesis upon a primer of RNA and the subsequent attachment of the second deoxyribonucleoside triphosphate.
The RNA-primed synthesis of DNA demonstrating the template function of the complementary strand of parental DNA.
The discontinuous polymerization of deoxyribonucleotides on the lagging strand; formation of Okazaki fragments during lagging strand DNA synthesis is illustrated. Okazaki fragments are 100 to 250 nucleotides long in eukaryotes, 1000 to 2000 nucleotides in prokaryotes.
Replication Exhibits Polarity
As has already been noted, DNA molecules are double stranded and the two strands are antiparallel. The replication of DNA in prokaryotes and eukaryotes occurs on both strands simultaneously. However, an enzyme capable of polymerizing DNA in the 3′ to 5′ direction does not exist in any organism, so that both of the newly replicated DNA strands cannot grow in the same direction simultaneously. Nevertheless, in bacteria the same enzyme does replicate both strands at the same time (in eukaryotes Pol ε and Pol δ catalyze leading and lagging strand synthesis; see Table 35–6. The single enzyme replicates one strand (“leading strand”) in a continuous manner in the 5′ to 3′ direction, with the same overall forward direction. It replicates the other strand (“lagging strand”) discontinuously while polymerizing the nucleotides in short spurts of 150 to 250 nucleotides, again in the 5′ to 3′ direction, but at the same time it faces toward the back end of the preceding RNA primer rather than toward the unreplicated portion. This process of semidiscontinuous DNA synthesis is shown diagrammatically in Figures 35–13 and 35–16.
Formation of Replication Bubbles
Replication of the circular bacterial chromosome, composed of roughly 5 × 106 bp of DNA proceeds from a single ori. This process is completed in about 30 minutes, a replication rate of 3 × 105 bp/min. The entire mammalian genome replicates in approximately 9 hours, the average period required for formation of a tetraploid genome from a diploid genome in a replicating cell. If a mammalian genome (3 × 109 bp) replicated at the same rate as bacteria (ie, 3 × 105 bp/min) from but a single ori, replication would take over 150 hours! Metazoan organisms get around this problem using two strategies. First, replication is bidirectional. Second, replication proceeds from multiple origins in each chromosome (a total of as many as 100 in humans). Thus, replication occurs in both directions along all of the chromosomes, and both strands are replicated simultaneously. This replication process generates “replication bubbles” (Figure 35–17).
The generation of “replication bubbles” during the process of DNA synthesis. The bidirectional replication and the proposed positions of unwinding proteins at the replication forks are depicted.
The multiple ori sites that serve as origins for DNA replication in eukaryotes are poorly defined except in a few animal viruses and in yeast. However, it is clear that initiation is regulated both spatially and temporally, since clusters of adjacent sites initiate replication synchronously. Replication firing, or DNA replication initiation at a replicator/ori, is influenced by a number of distinct properties of chromatin structure that are just beginning to be understood. It is clear, however, that there are more replicators and excess ORC than needed to replicate the mammalian genome within the time of a typical S-phase. Therefore, mechanisms for controlling the excess ORC-bound replicators must exist. Understanding the control of the formation and firing of replication complexes is one of the major challenges in this field.
During the replication of DNA, there must be a separation of the two strands to allow each to serve as a template by hydrogen bonding its nucleotide bases to the incoming deoxynucleoside triphosphate. The separation of the DNA strands is promoted by single strand DNA binding proteins (SSBs) in E coli, and a protein termed replication protein A (RPA) in eukaryotes. These molecules stabilize the single-stranded structure as the replication fork progresses. The stabilizing proteins bind cooperatively and stoichiometrically to the single strands without interfering with the abilities of the nucleotides to serve as templates (Figure 35–13). In addition to separating the two strands of the double helix, there must be an unwinding of the molecule (once every 10 nucleotide pairs) to allow strand separation. The hexameric DNA β protein complex unwinds DNA in E coli, whereas the hexameric MCM complex unwinds eukaryotic DNA. This unwinding happens in segments adjacent to the replication bubble. To counteract this unwinding, there are multiple “swivels” interspersed in the DNA molecules of all organisms. The swivel function is provided by specific enzymes that introduce “nicks” in one strand of the unwinding double helix, thereby allowing the unwinding process to proceed. The nicks are quickly resealed without requiring energy input, because of the formation of a high-energy covalent bond between the nicked phosphodiester backbone and the nicking-sealing enzyme. The nicking-resealing enzymes are called DNA topoisomerases. This process is depicted diagrammatically in Figure 35–18 and there compared with the ATP-dependent resealing carried out by the DNA ligases. Topoisomerases are also capable of unwinding supercoiled DNA. Supercoiled DNA is a higher-ordered structure occurring in circular DNA molecules wrapped around a core, as depicted in Figures 35–2 and 35–19.
Comparison of two types of nick-sealing reactions on DNA. The series of reactions at left is catalyzed by DNA topoisomerase I, that at right by DNA ligase; P, phosphate; R, ribose; A, adenine. (Slightly modified and reproduced, with permission, from Lehninger AL: Biochemistry, 2nd ed. Worth, 1975. Copyright © 1975 by Worth Publishers. Used, with permission, from W. H. Freeman and Company.)
Supercoiling of DNA. A left-handed toroidal (solenoidal) supercoil, at left, will convert to a right-handed interwound supercoil, at right, when the cylindric core is removed. Such a transition is analogous to that which occurs when nucleosomes are disrupted by the high salt extraction of histones from chromatin.
There exists in one species of animal viruses (retroviruses) a class of enzymes capable of synthesizing a single-stranded and then a dsDNA molecule from a single-stranded RNA template. This polymerase, termed RNA-dependent DNA polymerase, or “reverse transcriptase,” first synthesizes a DNA–RNA hybrid molecule utilizing the RNA genome as a template. A specific virus-encoded nuclease, RNase H, degrades the hybridized template RNA strand, and the remaining DNA strand in turn serves as a template to form a dsDNA molecule containing the information originally present in the RNA genome of the animal virus.
Reconstitution of Chromatin Structure
There is evidence that nuclear organization and chromatin structure are involved in determining the regulation and initiation of DNA synthesis. As noted above, the rate of polymerization in eukaryotic cells, which have chromatin and nucleosomes, is slower than that in prokaryotic cells, which lack canonical nucleosomes. It is also clear that chromatin structure must be re-formed after replication. Newly replicated DNA is rapidly assembled into nucleosomes, and the preexisting and newly assembled histone octamers are randomly distributed to each arm of the replication fork. These reactions are facilitated through the actions of histone chaperone proteins working in concert with chromatin assembly and remodeling complexes.
DNA Synthesis Occurs During the S Phase of the Cell Cycle
In animal cells, including human cells, the replication of the DNA genome occurs only at a specified time during the life span of the cell. This period is referred to as the synthetic or S phase. This is usually temporally separated from the mitotic, or M phase, by nonsynthetic periods referred to as gap 1 (G1) and gap 2 (G2) phases, occurring before and after the S phase, respectively (Figure 35–20). Among other things, the cell prepares for DNA synthesis in G1, and for mitosis in G2. The cell regulates DNA synthesis by allowing it to occur only once per cell cycle, and only during S-phase, in cells preparing to divide by a mitotic process.
Progress through the mammalian cell cycle is continuously monitored via multiple cell-cycle checkpoints. DNA, chromosome, and chromosome segregation integrity is continuously monitored throughout the cell cycle. If DNA damage is detected in either the G1 or the G2 phase of the cell cycle, if the genome is incompletely replicated, or if normal chromosome segregation machinery is incomplete (ie, a defective spindle), cells will not progress through the phase of the cycle in which defects are detected. In some cases, if the damage cannot be repaired, such cells undergo programmed cell death (apoptosis). Note that cells can reversibly leave the cell cycle during G1 entering a nonreplicative state termed G0 (not shown, but see Figure 9–8). When appropriate signals/conditions occur cells re-enter G1 and progress normally through the cell cycle as depicted.
All eukaryotic cells have gene products that govern the transition from one phase of the cell cycle to another. The cyclins are a family of proteins whose concentration increases and decreases at specific times, that is, “cycle” during the cell cycle—thus their name. The cyclins thus activate, at the appropriate time, different cyclin-dependent protein kinases (CDKs) that phosphorylate substrates essential for progression through the cell cycle (Figure 35–21). For example, cyclin D levels rise in late G1 phase and allow progression beyond the start (yeast) or restriction point (mammals), the point beyond which cells irrevocably proceed into the S or DNA synthesis phase.
Schematic illustration of the points during the mammalian cell cycle during which the indicated cyclins and cyclin-dependent kinases are activated. The thickness of the various colored lines is indicative of the extent of activity.
The D cyclins activate CDK4 and CDK6. These two kinases are also synthesized during G1 in cells undergoing active division. The D cyclins and CDK4 and CDK6 are nuclear proteins that assemble as a complex in late G1 phase. The cyclin-CDK complex is now an active serine-threonine protein kinase. One substrate for this kinase is the retinoblastoma (Rb) protein. Rb is a cell-cycle regulator because it binds to and inactivates a transcription factor (E2F) necessary for the transcription of certain genes (histone genes, DNA replication proteins, etc) needed for progression from G1 to S phase. The phosphorylation of Rb by CDK4 or CDK6 results in the release of E2F from Rb-mediated transcription repression—thus, gene transcription activation ensues and cell-cycle progression takes place.
Other cyclins and CDKs are involved in different aspects of cell-cycle progression (Table 35–7). Cyclin E and CDK2 form a complex in late G1. Cyclin E is rapidly degraded, and the released CDK2 then forms a complex with cyclin A. This sequence is necessary for the initiation of DNA synthesis in S phase. A complex between cyclin B and CDK1 is rate-limiting for the G2/M transition in eukaryotic cells.
TABLE 35–7Cyclins and Cyclin-Dependent Kinases Involved in Cell-Cycle Progression ||Download (.pdf) TABLE 35–7 Cyclins and Cyclin-Dependent Kinases Involved in Cell-Cycle Progression
|Cyclin ||Kinase ||Function |
|D ||CDK4, CDK6 ||Progression past restriction point at G1/S boundary |
|E, A ||CDK2 ||Initiation of DNA synthesis in early S phase |
|B ||CDK1 ||Transition from G2 to M |
Many of the cancer-causing viruses (oncoviruses) and cancer-inducing genes (oncogenes) are capable of alleviating or disrupting the apparent restriction that normally controls the entry of mammalian cells from G1 into the S phase. From the foregoing, one might have surmised that excessive production of a cyclin, loss of a specific CDK inhibitor (see below), or production or activation of a cyclin/CDK at an inappropriate time might result in abnormal or unrestrained cell division. In this context, it is noteworthy that the bcl oncogene associated with B-cell lymphoma appears to be the cyclin D1 gene. Similarly, the oncoproteins (or transforming proteins) produced by several DNA viruses target the Rb transcription repressor for inactivation, inducing cell division inappropriately, while inactivation of Rb, itself a tumor suppressor gene, leads to uncontrolled cell growth and tumor formation.
During the S phase, mammalian cells contain greater quantities of DNA polymerase than during the nonsynthetic phases of the cell cycle. Furthermore, those enzymes responsible for formation of the substrates for DNA synthesis—that is, deoxyribonucleoside triphosphates—are also increased in activity, and their expression drops following the synthetic phase until the reappearance of the signal for renewed DNA synthesis. During the S phase, the nuclear DNA is completely replicated once and only once. Once chromatin has been replicated, it is marked so as to prevent its further replication until it again passes through mitosis. This process is termed replication licensing. The molecular mechanisms for this phenomenon in human cells involves dissociation and/or cyclin-CDK phosphorylation and subsequent degradation of several origin binding proteins that play critical roles in replication complex formation. Consequently origins fire only once per cell cycle.
In general, a given pair of chromosomes will replicate simultaneously and within a fixed portion of the S phase upon every replication. On a chromosome, clusters of replication units replicate coordinately. The nature of the signals that regulate DNA synthesis at these levels is unknown, but the regulation does appear to be an intrinsic property of each individual chromosome that is mediated by the several replication origins contained therein.
All Organisms Contain Elaborate Evolutionarily Conserved Mechanisms to Repair Damaged DNA
Repair of damaged DNA is critical for maintaining genomic integrity and thereby preventing the propagation of mutations, either horizontally, that is DNA sequence changes in somatic cells, or vertically, where nonrepaired lesions are present in sperm or oocyte DNA and hence can be transmitted to progeny. DNA is subjected to a huge array of chemical, physical, and biological assaults on a daily basis (Table 35–8), hence efficient recognition and repair of DNA lesions is essential. Consequently, eukaryotic cells contain five major DNA repair pathways, each of which contain multiple, sometimes shared proteins; these DNA repair proteins typically have orthologues in prokaryotes. The mechanisms of DNA repair include nucleotide excision repair (NER); mismatch repair (MMR); base excision repair (BER); homologous recombination (HR); and nonhomologous end-joining (NHEJ) repair pathways (Figure 35–22). The experiment of testing the importance of many of these DNA repair proteins to human biology has been performed by nature—mutations in a large number of these genes lead to human disease (Table 35–9). Moreover, systematic gene-directed “knock-out” experiments (see Chapter 39) with laboratory mice have clearly ascribed critical gene integrity maintenance functions to these genes as well. In the mouse genetic studies, it was observed that indeed targeted mutations within these genes induce defects in DNA repair while often also dramatically increasing susceptibility to cancer.
TABLE 35–8Types of Damage to DNA ||Download (.pdf) TABLE 35–8 Types of Damage to DNA
I. Single-base alteration
B. Deamination of cytosine to uracil
C. Deamination of adenine to hypoxanthine
D. Alkylation of base
F. Base-analog incorporation
II. Two-base alteration
A. UV light-induced thymine-thymine (pyrimidine) dimer
B. Bifunctional alkylating agent cross-linkage
III. Chain breaks
A. Ionizing radiation
B. Radioactive disintegration of backbone element
C. Oxidative free radical formation
A. Between bases in same or opposite strands
B. Between DNA and protein molecules (eg, histones)
Mammals use multiple DNA repair pathways of variable accuracy to repair the myriad forms of DNA damage genomic DNA is subjected to. Listed are the major types of DNA damaging agents, the DNA lesions so formed (schematized and listed), the DNA repair pathway responsible for repairing the different lesions, and the relative fidelity of these pathways. (Modified, with permission, from: “DNA-Damage Response in Tissue-Specific and Cancer Stem Cells” Cell Stem Cell 8:16–29 (2011) copyright © 2011 Elsevier Inc.
TABLE 35–9Human Diseases of DNA Damage Repair ||Download (.pdf) TABLE 35–9 Human Diseases of DNA Damage Repair
Defective Nonhomologous End Joining Repair (NHEJ)
Severe combined immunodeficiency disease (SCID)
Radiation sensitive severe combined immunodeficiency disease (RS-SCID)
Defective Homologous Repair (HR)
AT-like disorder (ATLD)
Nijmegen breakage syndrome (NBS)
Bloom syndrome (BS)
Werner syndrome (WS)
Rothmund-Thomson syndrome (RTS)
Breast cancer suspectibility 1 and 2 (BRCA1, BRCA2)
Defective DNA Nucleotide Exicision Repair (NER)
Xeroderma pigmentosum (XP)
Cockayne syndrome (CS)
Defective DNA Base Excision Repair (BER)
MUTYH-associated polyposis (MAP)
Defective DNA Mismatch Repair (MMR)
Hereditary nonpolyposis colorectal cancer (HNPCC)
One of the most intensively studied mechanisms of DNA repair is the mechanism used to repair DNA double-strand breaks (DSBs); these will be discussed in some detail here. There are two pathways, HR and NHEJ, that eukaryotic cells utilize to remove DSBs. The choice between the two depends upon the phase of the cell cycle (Figures 35–20 and 35–21) and the exact type of DSB breaks to be repaired (Table 35–8). During the G0/G1 phases of the cell cycle, DSBs are corrected by the NHEJ pathway, whereas during S, G2, and M phases of the cell cycle HR is utilized. All steps of DNA damage repair are catalyzed by evolutionarily conserved molecules, which include DNA damage Sensors, Transducers, and damage repair Mediators. Collectively, these cascades of proteins participate in the cellular response to DNA damage. Importantly, the ultimate cellular outcomes of DNA damage and cellular attempts to repair DNA damage range from cell-cycle delay to allow for DNA repair, to cell-cycle arrest, to apoptosis or senescence (see Figure 35–23; and further detail below). The molecules involved in these complex and highly integrated processes range from damage-specific histone modifications (ie, dimethylated lysine 20 histone H4; H4K20me2) and incorporation of histone isotype variants such as histone H2AX into nucleosomes at the site of DNA damage (cf Table 35–1), poly ADP ribose polymerase, PARP, the MRN protein complex (Mre11-Rad50-NBS1 subunits); to DNA damage-activated kinase recognition/signaling proteins (ATM [ataxia telangiectasia, mutated] and ATM-related kinase, ATR, the multisubunit DNA-dependent protein kinase [DNA-PK and Ku70/80], and checkpoint kinases 1 and 2 [CHK1, CHK2]). These multiple kinases phosphorylate, and consequently modulate the activities of dozens of proteins, such as numerous DNA repair, checkpoint control, and cell-cycle control proteins like CDC25A, B, C, Wee1, p21, p16, and p19 (all Cyclin-CDK regulators [see Figure 9–8; and below]; various exo- and endonucleases; DNA single-strand-specific DNA-binding proteins [RPA]; PCNA and specific DNA polymerases [DNA pol delta, δ; and eta, η]). Several of these (types) of proteins/enzymes have been discussed above in the context of DNA replication. DNA repair and its relationship to cell cycle control are very active areas of research given their central roles in cell biology and potential for generating and preventing cancer.
The multistep mechanism of DNA double-strand break repair. Shown top to bottom are the proteins (protein complexes) that: identify DSBs in genomic DNA (sensors), transduce and amplify the recognized DNA damage (transducers and mediators), as well as the molecules that dictate the ultimate outcomes of the DNA damage response (effectors). Damaged DNA can be: (a) repaired directly (DNA repair), or, via p53-mediated pathways and depending upon the severity of DNA damage and p53-activated genes induced, (b), cells can be arrested in the cell cycle by p21/WAF1 the potent CDK–cyclin complex inhibitor to allow time for extensively damaged DNA to be repaired, or (c), and (d) if the extent of DNA damage is too great to repair, cells can either apotose or senesce; both of these processes prevent the cell containing such damaged DNA from ever dividing and hence inducing cancer or other deleterious biological outcomes. (Based on: “DNA-Damage Response in Tissue-Specific and Cancer Stem Cells” Cell Stem Cell 8:16–29 (2011) copyright © 2011 Elsevier Inc.)
DNA & Chromosome Integrity Is Monitored Throughout the Cell Cycle
Given the importance of normal DNA and chromosome function to survival, it is not surprising that eukaryotic cells have developed elaborate mechanisms to monitor the integrity of the genetic material. As detailed above, a number of complex multisubunit enzyme systems have evolved to repair damaged DNA at the nucleotide sequence level. Similarly, DNA mishaps at the chromosome level are also monitored and repaired. As shown in Figure 35–20, both DNA and chromosomal integrity are continuously monitored throughout the cell cycle. The four specific steps at which this monitoring occurs have been termed checkpoint controls. If problems are detected at any of these checkpoints, progression through the cycle is interrupted and transit through the cell cycle is halted until the damage is repaired. The molecular mechanisms underlying detection of DNA damage during the G1 and G2 phases of the cycle are understood better than those operative during S and M phases.
The tumor suppressor p53, a protein of apparent MW 53 kDa on SDS-PAGE, plays a key role in both G1 and G2 checkpoint control. Normally a very unstable protein, p53 is a DNA-binding transcription factor, one of a family of related proteins (ie, p53, p63, and p73) that is somehow stabilized in response to DNA damage, perhaps by direct p53-DNA interactions. Like the histones discussed above, p53 is subject to a panoply of regulatory PTMs, all of which likely modify its multiple biological activities. Increased levels of p53 activate transcription of an ensemble of genes that collectively serve to delay transit through the cycle. One of these induced proteins, p21, is a potent CDK–cyclin inhibitor (CKI) that is capable of efficiently inhibiting the action of all CDKs. Clearly, inhibition of CDKs will halt progression through the cell cycle (see Figures 35–19 and 35–20). If DNA damage is too extensive to repair, the affected cells undergo apoptosis (programmed cell death) in a p53-dependent fashion. In this case, p53 induces the activation of a collection of genes that induce apoptosis. Cells lacking functional p53 fail to undergo apoptosis in response to high levels of radiation or DNA-active chemotherapeutic agents. It may come as no surprise, then, that p53 is one of the most frequently mutated genes in human cancers (Chapter 56). Indeed recent genomic sequencing studies of multiple tumor DNA samples suggest that over 80% of human cancers carry p53 loss of function mutations. Additional research into the mechanisms of checkpoint control will prove invaluable for the development of effective anticancer therapeutic options.