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Simultaneous disruption of two DNA polymerases, Pol? and Pol?, in Avian DT40 cells unmasks the role of Pol? in cellular response to various DNA lesions.

ABSTRACT: Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase ?, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ? by generating POL?(-/-)/POL?(-/-) cells from the chicken DT40 cell line. POL?(-/-) cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that Pol? plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POL?(-/-)/POL?(-/-) cells shows that, unexpectedly, the loss of Pol? significantly rescued all mutant phenotypes of POL?(-/-) cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, Pol? contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells.

SUBMITTER: Hirota K

PROVIDER: S-EPMC2951353 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Simultaneous disruption of two DNA polymerases, Polη and Polζ, in Avian DT40 cells unmasks the role of Polη in cellular response to various DNA lesions.

Hirota Kouji K Sonoda Eiichiro E Kawamoto Takuo T Motegi Akira A Masutani Chikahide C Hanaoka Fumio F Szüts Dávid D Iwai Shigenori S Sale Julian E JE Lehmann Alan A Takeda Shunichi S

PLoS genetics 20101007 10

Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase η, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase ζ by generating POLη(-/-)/POLζ(-/-) cells from ...[more]

PMID: 20949111

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Project description:Escherichia coli has five DNA polymerases, one of which, the low-fidelity Pol IV or DinB, is required for stress-induced mutagenesis in the well-studied Lac frameshift-reversion assay. Although normally present at approximately 200 molecules per cell, Pol IV is recruited to acts of DNA double-strand-break repair, and causes mutagenesis, only when at least two cellular stress responses are activated: the SOS DNA-damage response, which upregulates DinB approximately 10-fold, and the RpoS-controlled general-stress response, which upregulates Pol IV about 2-fold. DNA Pol III was also implicated but its role in mutagenesis was unclear. We sought in vivo evidence on the presence and interactions of multiple DNA polymerases during stress-induced mutagenesis. Using multiply mutant strains, we provide evidence of competition of DNA Pols I, II and III with Pol IV, implying that they are all present at sites of stress-induced mutagenesis. Previous data indicate that Pol V is also present. We show that the interactions of Pols I, II and III with Pol IV result neither from, first, induction of the SOS response when particular DNA polymerases are removed, nor second, from proofreading of DNA Pol IV errors by the editing functions of Pol I or Pol III. Third, we provide evidence that Pol III itself does not assist with but rather inhibits Pol IV-dependent mutagenesis. The data support the remaining hypothesis that during the acts of DNA double-strand-break (DSB) repair, shown previously to underlie stress-induced mutagenesis in the Lac system, there is competition of DNA polymerases I, II and III with DNA Pol IV for action at the primer terminus. Up-regulation of Pol IV, and possibly other stress-response-controlled factor(s), tilt the competition in favor of error-prone Pol IV at the expense of more accurate polymerases, thus producing stress-induced mutations. This mutagenesis assay reveals the DNA polymerases operating in DSB repair during stress and also provides a sensitive indicator for DNA polymerase competition and choice in vivo.

Project description:BackgroundEvolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved.ResultsWe performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase epsilon consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol epsilon evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol epsilon parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol epsilon, and the other one to the common ancestor of Pol alpha, Pol delta, and Pol zeta. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polepsilon shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases.ConclusionEvolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We hypothesize that the archaeal ancestor of eukaryotes encoded three DNA polymerases, namely, two distinct B-family polymerases and a D-family polymerase all of which contributed to the evolution of the eukaryotic replication machinery. The Zn-finger might have been acquired from PolD by the B-family form that gave rise to Pol epsilon prior to or in the course of eukaryogenesis, and subsequently, was captured by the ancestor of the other B-family eukaryotic polymerases. The inactivated polymerase-exonuclease module of Pol epsilon might have evolved by fusion with a distinct polymerase, rather than by duplication of the active module of Pol epsilon, and is likely to play an important role in the assembly of eukaryotic replication and repair complexes.

Dataset Information

Simultaneous disruption of two DNA polymerases, Pol? and Pol?, in Avian DT40 cells unmasks the role of Pol? in cellular response to various DNA lesions.

Publications

Simultaneous disruption of two DNA polymerases, Polη and Polζ, in Avian DT40 cells unmasks the role of Polη in cellular response to various DNA lesions.

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