The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small, modest changes in structure including mismatched bases and simple methylation events to oxidized bases, intra- and interstrand DNA crosslinks, DNA double strand breaks and protein-DNA adducts. Pathways required for the repair of these lesions include mismatch repair, base excision repair, nucleotide excision repair, and the homology directed repair/Fanconi anemia pathway. Each of these pathways contributes to genetic stability, and mutations in genes encoding proteins involved in these pathways have been demonstrated to promote genetic instability and cancer. In fact, it has been suggested all cancers display defects in DNA repair. It has also been demonstrated that the ability of cancer cells to repair therapeutically induced DNA damage impacts therapeutic efficacy. This has led to targeting DNA repair pathways and proteins to develop anti-cancer agents that will increase sensitivity to traditional chemotherapeutics. While initial studies languished and were plagued by a lack of specificity and a defined mechanism of action, more recent approaches to exploit synthetic lethal interaction and develop high affinity chemical inhibitors have proven considerably more effective. In this review we will highlight recent advances and discuss previous failures in targeting DNA repair to pave the way for future DNA repair targeted agents and their use in cancer therapy.

npg Repair of bulky DNA adducts by the nucleotide excision repair (NER) pathway is one of the more versatile DNA repair pathways for the removal of DNA lesions. There are two subsets of the NER pathway, global genomic-NER (GG-NER) and transcription-coupled NER (TC-NER), which differ only in the step involving recognition of the DNA lesion. Following recognition of the damage, the sub-pathways then converge for the incision/excision steps and subsequent gap filling and ligation steps. This review will focus on the GGR sub-pathway of NER, while the TCR sub-pathway will be covered in another article in this issue. The ability of the NER pathway to repair a wide array of adducts stems, in part, from the mechanisms involved in the initial recognition step of the damaged DNA and results in NER impacting an equally wide array of human physiological responses and events. In this review, the impact of NER on carcinogenesis, neurological function, sensitivity to environmental factors and sensitivity to cancer therapeutics will be discussed. The knowledge generated in our understanding of the NER pathway over the past 40 years has resulted from advances in the fields of animal model systems, mammalian genetics and in vitro biochemistry, as well as from reconstitution studies and structural analyses of the proteins and enzymes that participate in this pathway. Each of these avenues of research has contributed significantly to our understanding of how the NER pathway works and how alterations in NER activity, both positive and negative, influence human biology.

Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis fromanupstre fragment to generate a nick, and then ligation. (4)(5)(6) has prompted several proposed roles for the latter enzyme in DNA replication (7)(8)(9)(10). While the viral model has clarified priming and elongation steps in the replication fork, the exact steps involved in completion of lagging-strand synthesis are not yet clear. Simian virus 40 reconstitution reactions (11-13) indicated roles for the mammalian 5'-to-3' exonuclease, a nuclear DNA polymerase, DNA ligase, and possibly RNase H type I (RNase HI) in joining of nascent segments of the lagging strand. DNA ligase I, rather than III, was shown to be required (12, 13). The specific function of RNase HI and the cleavage mechanism involved remained to be determined.In Escherichia coli, initiator RNA of Okazaki fragments is thought to be removed by the 5'-to-3' exonuclease of DNA polymerase I, although a role for RNase H cannot be totally excluded (14). Double strand-specific 5'-to-3' exonucleases, which appear to be functional homologs of each other, have been isolated from human cells, mice, and calf (11-13, 15, 16). The calf nuclease displayed an endonuclease function that cleaves the unannealed 5' tail of a primer on a template, in the presence of a directly adjacent upstream primer (17). This very specific function is also exhibited by E. coli DNA polymerase 1 (18), which strongly suggests that the bacterial and mammalian enzymes are also functional homologs.Mammalian RNase H enzymes have been identified and characterized in detail. RNase H enzymes can be placed into one of two classes (I and II) based on subunit structure, peptide molecular mass, and the ability to use manganese as a cofactor (19,20). They have been postulated to be involved in DNA replication and transcription (21), but no specific roles have been established for either class.We had shown (22) that the calf 5'-to-3' exonuclease could work with calf DNA polymerases a, 8, or e and DNA ligase I to join two DNA primers annealed to a template with a four-base gap. In addition, we have demonstrated that 5'-to-3' exonuclease activity is stimulated by synthesis from an upstream primer and that the stimulation is the result of the formation of a nick between the two primers, which is a preferred substrate for the exonuclease (17). These results suggest that a nick translation process occurs prior to the joining reaction catalyzed by DNA ligase I. Goulian et al. (15) previously showed that a combination of RNase HI and 5'-to-3' exonuclease can remove initiator RNA from primertemplates in vitro. Here we define the specific reactions by which these two nucleases can remove initiator RNA in a model Okazaki fragment system before polymerization can make the substrate for ligation.MATERIALS AND METHODS Protein Purification. DNA polymerase E, DNA ligase I, a...

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Restarting stalled replication forks partly depends on the breakinduced recombination pathway, in which a DNA doublestranded break (DSB) is created on the stalled replication fork to initiate the downstream recombination cascades. Singlestranded DNA gaps accumulating on stalled replication forks are potential targets for endonucleases to generate DSBs. However, it is unclear how this process is executed and which nucleases are involved in eukaryotic cells. Here, we identify a novel gap endonuclease (GEN) activity of human flap endonuclease 1 (FEN-1), critical in resolving stalled replication fork. In response to replication arrest, FEN-1 interacts specifically with Werner syndrome protein for efficient fork cleavage. Replication protein A facilitates FEN-1 interaction with DNA bubble structures. Human FEN-1, but not the GEN-deficient mutant, E178A, was shown to rescue the defect in resistance to UV and camptothecin in a yeast FEN-1 null mutant.