DNA Polymerases

Four members, which are stongly implicated in bypassing DNA lesions that block replication by the B family replicative DNA polymerasesThe major role of Y-family DNA polymerases is to carry TLS past damaged bases

Pol k participates in bypass of several bulky DNA lesions. Study by Ogi et al, indicates that pol k has a function traditionally associated with B family polymerases, that is filling gaps during NERPol K can replicate past several bulky adducts in DNA in vitro and cellular studies have shown a role for pol K in TLS past lesions such as benzo (a) pyrene adductsHowever, pol k differs from the three other Y-family human polymerases in its localization pattern within the nucleusPrevious work has shown that DNA polymerases eta and iota are localised in replication factories during S phase, where they colocalise one-to-one with PCNA. Cells with factories containing these polymerases accumulate after treatment with DNA damaging agents because replication forks are stalled at sites of damage. We now show that DNA polymerase kappa (pol(kappa)) has a different localisation pattern. Although, like the other Y-family polymerases, it is exclusively localised in the nucleus, pol(kappa) is found in replication foci in only a small proportion of S-phase cells. It does not colocalise in those foci with proliferating cell nuclear antigen (PCNA) in the majority of cells. This reduced number of cells with pol(kappa) foci, when compared with those containing pol(eta) foci, is observed both in untreated cells and in cells treated with hydroxyurea, UV irradiation or benzo[a]pyrene. The C-terminal 97 amino acids of pol(kappa)are sufficient for this limited localisation into nuclear foci, and include a C2HC zinc finger, bipartite nuclear localisation signal and putative PCNA binding siteFuthermore, several independent reports have shown that complete disruption of the Polk gene in mouse and chicken cells causes significant UV sensitivity, even though, in vitro, pol k cannot on its own bypass either of the major UV lesions.

Previous work has shown that DNA polymerases eta and iota are localised in replication factories during S phase, where they colocalise one-to-one with PCNA. Cells with factories containing these polymerases accumulate after treatment with DNA damaging agents because replication forks are stalled at sites of damage. We now show that DNA polymerase kappa (pol(kappa)) has a different localisation pattern. Although, like the other Y-family polymerases, it is exclusively localised in the nucleus, pol(kappa) is found in replication foci in only a small proportion of S-phase cells. It does not colocalise in those foci with proliferating cell nuclear antigen (PCNA) in the majority of cells. This reduced number of cells with pol(kappa) foci, when compared with those containing pol(eta) foci, is observed both in untreated cells and in cells treated with hydroxyurea, UV irradiation or benzo[a]pyrene. The C-terminal 97 amino acids of pol(kappa)are sufficient for this limited localisation into nuclear foci, and include a C2HC zinc finger, bipartite nuclear localisation signal and putative PCNA binding siteFuthermore, several independent reports have shown that complete disruption of the Polk gene in mouse and chicken cells causes significant UV sensitivity, even though, in vitro, pol k cannot on its own bypass either of the major UV lesions.

The previous findings prompted us to consder an alternative role for pol k that would explains its UV sensitivity

NER is the process by which lesions that distort DNA helix geometry are removed from DNA and replaced with an undamaged nucleotide. NER was discovereed over 40 years ago and has since been fully reconstituted with purified proteins. NER can be conducted in a global, genome-wide manner (GGR), or as transcription-coupled repair (TCR) NER consists of a general pathway termed global genome repair (GGR) that removes lesions from the entire genome and a specialized pathway referred to as transcription-coupled repair (TCR). Repair by TCR is confined to DNA lesions in the transcribed strand of transcriptionally active genes and strictly depends on ongoing transcription by RNA polymerase II (RNAPolII) Examination of the repair kinetics of structurally different DNA lesions has disclosed that TCR functions as an efficient repair system for transcription-blocking lesions that are poorly repaired by GGR. In the case of UV-induced photolesions, repair of 6-4PP is fast throughout the genomeand is dominated by GGR. In contrast, GGR of CPD is relatively slow, but TCR causes accelerated removal of this lesion from the transcribed strand of expressed genes (van Hoffen et al., 1995).Humans cells exhibit both GGR and TCR of CPD resulting in the preferential repair of CPd in the transcribed strand of expressed genes.

Examination of the repair kinetics of structurally different DNA lesions has disclosed that TCR functions as an efficient repair system for transcription-blocking lesions that are poorly repaired by GGR. In the case of UV-induced photolesions, repair of 6-4PP is fast throughout the genomeand is dominated by GGR. In contrast, GGR of CPD is relatively slow, but TCR causes accelerated removal of this lesion from the transcribed strand of expressed genes (van Hoffen et al., 1995). The XPC-hHR23B heterodimer is the principal damage recognition factor in GGR and is strictly required for the recruitment of all following NER proteins to the damaged DNAAmong the various complementation groups, XP-C is unique, as only GGR is compromised in this group

Repair by TCR is confined to DNA lesions in the transcribed strand of transcriptionally active genes and strictly depends on ongoing transcription by RNA polymerase II (RNAPolII).TCR is facilitated by RNA polymerase stalled at sites of damage, which helps recruit the repair machinery to the site of the damage.Examination of the repair kinetics of structurally different DNA lesions has disclosed that TCR functions as an efficient repair system for transcription-blocking lesions that are poorly repaired by GGR. Examination of the repair kinetics of structurally different DNA lesions has disclosed that TCR functions as an efficient repair system for transcription-blocking lesions that are poorly repaired by GGR. In the case of UV-induced photolesions, repair of 6-4PP is fast throughout the genomeand is dominated by GGR. In contrast, GGR of CPD is relatively slow, but TCR causes accelerated removal of this lesion from the transcribed strand of expressed genes (van Hoffen et al., 1995).In contrast to ERCC1-XPF, the recruitment of XPG to the incision complex does not depend on functional XPA protein. However, its recruitment is apparently insufficient to perform 3 incision, since UV-irradiated XP-A cells are completely devoid of incision activity. Most likely, incision by XPG is activated by the XPA protein in cooperation with RPA, since the latter has been shown to be essential for the formation of dual incision in NER (Mu et al., 1996; de Laat et al., 1998).

TLS alleviates stalinig of replication forks that may lead to mutagenesis and/or cell death.

Pol n bypasses sunlight induced cyclobutante pyrimidine dimers that inhibit the replicative polymerases alpha, delta, episilion

TLS alleviates stalinig of replication forks that may lead to mutagenesis and/or cell death.

A defect in human pol n in xeroderma pigmentosum variant patients results in increased UV light-induced cytotoxicity and mutagenesis, and greatly increased susceptibility to sunlight-induced skin cancer

Replicative polymerases, are stalled by DNA lesions