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- Title
Expression and Activity of Human DNA Polymerase h in Escherichia coli.
- Authors
Grúz, Petr; Takehiko Nohmi
- Abstract
DNA polymerase h (hPolŋ) is a key protein in translesion DNA synthesis (TLS) in human cells. Its primary function is the error free replication through UV-induced TT cyclobutane dimers which present a barrier to DNA synthesis by other eukaryotic replicative polymerases. hPolŋ defects underlie the genetic disease xeroderma pigmentosum variant (XPV) characterized by higher susceptibility to UV-light induced skin cancers due to erroneous replication of the UV adducts. However, hPolŋ is also a very low fidelity enzyme when copying undamaged DNA or DNA with other adducts and is actively recruited during the somatic hypermutation of the immunoglobulin genes. Here, we demonstrate that hPolŋ restores partially the mutability and completely the survival of a UV non-mutable umuDC-deletion mutant of Escherichia coli after UVB irradiation. We chose UVB instead of UVC as a radiation source because UVB is a major cause of human skin cancer induced by sunlight. The umuDC genes encode endogenous TLS DNA polymerase V. The catalytic core lacking the C-terminal part of hPolŋ was even more biologically active than the full size protein and its activity was further enhanced by attaching the prokaryotic b-subunit binding motif to it. The mutagenicity and survival effects were enhanced upon the induction of hPolŋ expression and its catalytically inactive variant was unable to promote any mutagenesis. This suggests that hPolŋ directly participates in the replication of damaged DNA in the prokaryotic bacteria. To demonstrate that our system can be useful in studying different variants of hPolh in vivo we have constructed 4 amino acid substitution mutants with altered geometry of the catalytic site analyzed previously biochemically and conrmed their altered abilities to promote mutagenesis and survival after UVB irradiation. This study paves a way to generate a variety of useful derivatives of hPolŋ in prokaryotic systems.
- Subjects
ESCHERICHIA coli; DNA polymerases; ESCHERICHIA coli DNA; CYCLOBUTANE derivatives; CANCER; DNA replication; MUTAGENESIS
- Publication
Genes & Environment, 2013, Vol 35, Issue 1, p10
- ISSN
1880-7046
- Publication type
Article
- DOI
10.3123/jemsge.35.10