DNA Repair

The outcome of genomic damage is dependent on the type of DNA lesions and the ability of the cell to repair DNA efficiently. A limited extent of DNA damage usually triggers apoptosis to eliminate the damaged cells or arrests the cell cycle to have more time to repair the damaged DNA. If the damage of DNA surpasses the DNA-repair capacity of the cell, genomic mutations or instability will be inevitable.

Damage to the genome can block the movement of RNA polymerase II (RNAPII) on the transcribed strand of active genes and consequently inactivate the damaged genes.20 Such damage includes ROS-induced 8-oxo-dG formation21 and UV-induced cyclobutane pyrimidine dimers.22 The stalled RNAPII on the damage site recruits Cockayne's Syndrome A (CSA) and CSB proteins23 and initiates a DNA repair process, the so-called transcription-coupled repair (TCR) through the recruitment of other DNA repair factors, such as TFIIH, XPG, XPA, RPA, and ERCC1, to the damaged site. During the initiation of TCR, the RNAPII is phosphorylated and modified by polyubiquitin chains that target RNAPII into the proteasomal degradation pathway.24 The transcription-coupled repair was considered a specific subpathway of the nucleotide excision repair (NER) pathway and base excision repair (BER) pathway.25

DNA damage may also interfere with the replication of DNA by blocking the activity of replicative DNA polymerase-5/e (pol5/e).26 The new types of DNA polymerase, pol^ and polK will act as alternative polymerases to replicate the DNA around the damaged sites.2728 Since these DNA polymerases have more flexible base-pairing properties, the replicated DNA contains a higher rate of error, which may be particularly relevant for the damage-induced point mutations seen in oncogenesis.2829

Based on the types of DNA damage, eukaryotic cells developed a number of DNA repair systems that are conserved in prokaryotic cells.30 In mammalian cells, at least three main types of DNA repair systems—excision repair, recombinational repair, and DNA post-replication repair (PRR)—function to repair damaged DNA. In the system of excision repair, there are three subtypes of repair mechanisms: nucleotide-excision repair (NER), base-excision repair (BER), and mismatch repair (MMR). The recombinational repair includes homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). Although each type or subtype of DNA repair systems requires a unique subset of DNA repairing factors, a certain degree of overlapping in the damage repair pathways has been noted under many circumstances.

The NER repairs DNA by removing bulky DNA adducts that distort the DNA helix structure and interfere with the base pairing that affects transcription and normal replication. BER, on the other hand, predominantly deals with small chemical modifications or alterations of bases. Certain incorrect base pairs that do not follow the Watson-Crick pairing are corrected by MMR. When DNA double strand breaks or DNA interstrand cross-links occur, cells employ HRR or NHEJ to repair such damages. HRR repairs damage during DNA replication in S and early G2 phases of the cell cycle by using the second sequence of sister chromatid to align the DNA breaks. In contrast, NHEJ repairs damaged DNA by a less-accurate DNA repairing process in the G1 phase of the cell cycle where a second copy is not available.20

The DNA PRR has been studied mainly in prokaryotic cells or yeast. Little is known about the mechanisms of PRR in mammalian cells. PRR converts single-stranded gaps of DNA damage into large-molecular-weight DNA without eliminating the replication-blocking lesions. Two modes of PRR have been suggested. The error-free PRR repairs damage through the use of undamaged sister chromatid as template. The error-prone PRR, however, uses specific DNA polymerases that are able to read through sites of DNA damage. The initiation of PRR in yeast requires RadlS, a single stranded DNA binding protein, and Rad6/UbcH1, an ubiquitin-conjugating enzyme.31 Human Rad6/UbcH1 has been demonstrated to be able to rescue yeast rad6 sensitivity to 8-

methoxypsoralen plus UVA (32), indicating a possible evolutional conservation of PRR within eukaryotes. The key regulatory mechanism of PRR is the modifications of PRR components by ubiquitin and SUMO. A recent study by Hoege et al.33 indicated that Rad6/Rad18-dependent monoubiquitination of PCNA resulted in either the recruitment of specific DNA polymerases for error-prone PRR or polyubiquitination of PCNA by Rad5 and Mms2/Ubc13 complex to activate error-free PRR. DNA Damage Response Signals

In general, different types of DNA damage activate unique signaling pathways. For instance, damage that triggers HRR usually activates ATM, BRCA1, and BRCA2, whereas damage that induces NHEJ predominantly activates DNA-PK.34 Nevertheless, the various forms of DNA damage share common downstream signal transduction pathways, and cross-talk among different signaling pathways can occur even in the very early stages of DNA damage responses. One of the most important DNA damage responses is the activation of checkpoints that causes cell cycle arrest at specific phases and/or cell apoptosis. Extensive studies have been made in the last decade that revealed a number of checkpoint proteins that can sense various types of DNA lesions. These checkpoint proteins include ATM, ATR, RAD17, RAD9, BRCA1/2, checkpoint protein 1 (Chk1), and Chk2.

The most established signaling pathway of DNA damage is the cellular response to DNA double-strand breaks (DSB)34 resulting from attacks by exogenous agents, such as ionizing radiation and ROS. In addition, DSB can also be derived from formation of improper DNA replication forks due to DNA single-strand breaks. DSB can be repairedby two major repair pathways: HRR and NHEJ systems. At the initiation of HRR, the DNA ends are recognized and bound by a complex containing RAD51, ATM, and BRCA1 or BRCA2 proteins.35,36 Such recruitment of ATM to the DNA damage sites activates the kinase activity of ATM that consequently phosphorylates p53, Mdm2, Chk2, BRCA1, NBS1, and c-Abl. In NHEJ, the free DNA ends are bound with the Ku subunits of DNA-PK, another PIKK family member. DNA-PK consists of a heterodimeric DNA binding subunit (Ku70 and Ku80) and a catalytic serine/threonine kinase subunit whose activity is activated by the association with DNA. Activated DNA-PK is able to

phosphorylate p53 or MDM2, ' although this conclusion remains controversial.

One of the most common outcomes of DNA damage response is cell cycle arrest through the activation of checkpoint pathways, which is considered a critical event for the cells to have more time to repair their damaged DNA. A number of checkpoints have been shown to be able to prevent the replication of damaged DNA or segregation of damaged chromosomes. The downstream targets of DNA damage-activated kinases, ATM and DNA-PK, are pivotal in executing the checkpoint signal transduction. Phosphorylation and activation of p53 by ATM or DNA-PK have been demonstrated as important processes of the G1 cell cycle checkpoint. The posttranslational modifications of p53 not only increase the stability of p53, but also enhance the transcriptional activity of p53 on its target genes, especially the G1 cyclin-dependent kinase inhibitor p21waf1.40 The p21waf1 suppresses cyclin E- and cyclin A-associated Cdk2 kinase activity and, thereby, blocks the cell cycle transition from G1 to S phase. The G2/M phase cell cycle arrest may be mainly mediated by the activation of checkpoint protein kinases Chk1 and

Chk2. Activated Chk1 or Chk2 phosphorylates a conserved site, Ser216, on the Cdc25C protein, a dual specific phosphatase that maintains the progress of the G2/M phase by dephosphorylating Tyr15 of cyclin-dependent kinase Cdc2. This phosphorylated Cdc25C is functionally inactive due to the binding of 14-3-3 protein that sequesters Cdc25C in cytoplasm and, therefore, prevents its phosphatase activity toward Tyr15 of Cdc2, an essential kinase for G2/M phase cell cycle transition.41

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