finally reestablishing the original DNA content using pathway-specific polymerases and ligases. Base excision repair acts during all cell cycle stages and is often responsible for correcting damages that arise spontaneously due to the inherent instability of DNA or due to exposure to intercalating (i.e., anticancer) agents and environmental mutagens that generate free radicals. Nucleotide excision repair largely acts independently of the cell cycle to remove bulky DNA adducts, such as UV-induced cyclobutane pyrimidine dimers, DNA cross-links, and certain oxidative base modifications. Mismatch repair acts to remove not only mismatches, but also small insertions or deletions that arise as replication errors or that arise during recombination.

Direct reversal of damage is a highly specialized repair mechanism. In humans, only the MGMT (O6-methyl- guanine-DNA methyltransferase) protein is known to function by this repair mechanism and irreversibly accepts the methyl group directly from the modified base.

Recombinational repair is required to repair DSB and is thus especially important in response to IR. DSBs are among the most harmful of lesions because they affect both strands of the double helix, meaning that one strand of DNA cannot act as a template for the repair of the other. There are two forms of DSBs: (1) twoended breaks, generated primarily by direct attack on DNA by a physical or chemical mutagen such as IR, and

(2) one-ended breaks, created when the replication fork collides into an unrepaired DNA single-strand break. One-ended breaks appear to be resolved strictly by classical homologous recombination. Two-ended breaks are repaired by three major repair mechanisms: (1) homologous recombination, (2) single-strand annealing, and (3) nonhomologous end-joining. Homologous recombination usually takes place after DNA replication (i.e., during S or G2 phase of the cell cycle) and is largely errorfree. An undamaged, homologous molecule such as a sister chromatid provides the repair template. On the other hand, single-strand annealing is an error-prone repair system. Homologous sequences (usually repetitive elements) on either side of the DSB are aligned followed by the deletion of the intermediate noncomplementary sequence. Nonhomologous end joining (NHEJ) is the major DSB repair pathway that takes place during the G1 phase of the cell cycle. This pathway is prone to errors because it fuses together two ends of a DSB.

2. DNA DAMAGE RESPONSE

An important question in cell biology is how the cell detects DNA damage. The cellular response to genotoxic stress can be envisioned as a highly conserved signal transduction cascade: the DNA damage response (DDR) (Figure 8-1). Sensor proteins are the first to detect DNA damage and replication stress. They then

RAD17

RPA

HUS1

RAD1

RAD9

ATR ATM

Chk1

2.2. Transducers

Various kinases of the phosphoino- sitide-3-kinase-related protein kinase (PIKK) family, including ataxia-telang- iectasia mutated (ATM) kinase (the gene altered in this recessive human genomic instability syndrome), ATMand Rad3-related (ATR) kinase, and

DNA-dependent protein kinase (DNA-PK), constitute the primary transducers of the DNA damage response. Within minutes of the DSB formation, ATM is recruited to the foci and activated. Active phosphorylated ATM remains stable for many hours. Unlike ATM, the ATR gene and its canonical substrate, Chk1, are essential in mice, underscoring their important role in normal cell growth. The ATR pathway is normally activated by stalled replication forks during DNA replication and thus plays an essential role in maintaining genome integrity during S phase. UV light, single-strand DNA, and presumably all chemical agents that give rise to stalled DNA replication forks also strongly activate the ATR pathway.

ATR is recruited by the single-strand DNA-RPA complex that also recruits and activates Rad17 and the proliferating cell nuclear antigen (PCNA)–related 911 (Rad9- Rad1-Hus1) complex. ATR phosphorylation of Rad17 and 911 is important for downstream signaling. It is not yet clear how ATR is activated when recruited to singlestrand DNA lesions, although both the 911 complex and TopBP1, another protein in the complex, have been

and has become a standard marker for such damage. Although unnecessary to promote the initial steps of the repair process, this modification is needed to concentrate repair machineries along the DNA lesions and to recruit chromatin modifiers such as complexes INO80, Swr1, and NuA4, which relaxes the chromatin structure surrounding the DNA lesion.

PP2A dephosphorylation of H2AX has been recently shown to be significant in turning off the damage response. Other H2A modifications have been identified as signals for general damage or stress, whereas others play roles in distinct repair pathways. Specifically, H2A phosphorylation of S122 and S129 is required to repair DSBs either by homologous recombination or NHEJ pathways. Other residues have even more specific roles: T126 is important for homologous recombination but dispensable for NHEJ, whereas S2 and K127 are critical for NHEJ but have no role in homologous recombination. These data indicate that both the type of damage and selected repair pathway are marked by specific H2A modifications, creating a unique histone code for each type of damage and repair.

Another covalent histone modification implicated in the DNA damage response is the methylation of histone H3 at lysine 79 (H3K79me) by the histone methyltransferase Dot1. Unlike γH2AX, DNA damage does not induce H3K79me but is constitutively present on chromatin. Otherwise, DNA damage would increase the accessibility of methylated H3K79me, allowing 53BP1 to instead act during the early sensing step. Similarly, H4K20 plays an analogous role in recruiting Crb2.

Histone acetylation not only functions in protein recruitment, but also acts to relax the chromatin structure and therefore facilitates access of DSB repair proteins such as 53BP1, BRCA1, and Rad51 to the lesion. The acetylation status of histones is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), and some of these HATs and HDACs are recruited to the lesion.

In addition to covalent histone modifications mentioned previously, chromatin is directly manipulated by adenosine triphosphate (ATP)–dependent chromatin remodeling complexes. These complexes use energy from ATP hydrolysis to facilitate chromatin remodeling through nucleosome sliding, nucleosome disruption, and exchange of histone components. Although the roles of many ATP-dependent remodeling complexes were first identified in transcriptional regulation, it has recently been shown that some of these complexes (e.g., INO80, RSC, SWI/SNF, and SWR-C) are also recruited for DNA repair.

5. CELL CYCLE CHECKPOINT REGULATION

One of the primary responses to DNA damage besides stimulation of DNA repair is the activation of cell cycle checkpoints. Cell cycle checkpoints are regulatory pathways that control the order and timing of cell cycle transitions and ensure that critical processes at each phase of the cell cycle, such as DNA replication and chromosome segregation, are completed with high fidelity before progressing to the next phase. A timely cell cycle progression results in the correct transmission of genetic information from parent to daughter cells. When stimulated with suitable growth factors, quiescent cells leave the cell cycle’s resting phase, called gap 0 (G0), and enter gap 1 (G1) phase, then segue to DNA replication or synthesis

(S) phase, which is followed by a second gap (G2) phase, and finally on to cell division or mitosis (M). DNA damage effect on cell cycle is mainly seen in three checkpoints: (1) G1/S (G1), (2) intra-S phase, and (3) G2/M. When DNA damage is sensed, cells arrest the cell cycle at these specific phases by activating the appropriate DNA damage checkpoint(s). For instance, on perturbation of DNA replication by normal, stalled replication forks or by drugs that interfere with DNA synthesis, cells activate the checkpoint that arrests the cell cycle at G2/M transition until DNA replication is complete. Checkpoint pathways also induce transcription of genes that contribute to the repair and its quality control.

Checkpoint activation is mediated by transcriptional and post-transcriptional modifications of proteins that regulate the cell cycle. Such regulation is carried out by oscillations in cycling-dependent kinases (Cdks), which are positively regulated by cyclins (cdk-cyclin complexes) and by dephosphorylation (mediated by the dual specificity Cdc25 phosphatase family, including Cdc25A, Cdc25B, and Cdc25C); Cdks are negatively regulated by Cdk inhibitors and Wee1 and Mik1 kinase-dependent phosphorylation within the ATP-binding domain. The cdk-cyclin complexes influencing G1 progression and the G1/S checkpoint are primarily Cdk4-cyclin D, Cdk6cyclin D, and Cdk2-cyclin E, whereas Cdk2-cyclin E complexes normally promote the G1/S transition.

Progression into S phase, and transition from G2 into M, is regulated by Cdk2/cyclin A and Cdk1/cyclin B, respectively. During the DNA damage response, activation of ATM/ATR and Chk1 and Chk2 kinases leads to phosphorylation of all three Cdc25 phosphatases. Chk1 or Chk2 phosphorylation of Cdc25 leads to inhibitory sequestration of Cdc25C by 14–3-3 proteins and ubiquitin-mediated proteolysis of Cdc25A. Cdc25A acts earlier in the cell cycle than Cdc25B and Cdc25C, is thought to be important for maximal Cdk