Highly Frequent Frameshift DNA Synthesis by Human DNA Polymerase μ

111

145

DNA polymerase(Pol ) is a member of the Pol X family having properties in common with several other mammalian DNA polymerases. To obtain clues to possible functions in vivo, we have determined the fidelity of DNA synthesis by human Pol . The results indicate that the average single-base deletion error rate of Pol is higher than those of other mammalian polymerases. In fact, unlike other DNA polymerases, Pol generates single-base deletions at average rates that substantially exceed base substitution rates. Moreover, the sequence specificity for single-base deletions made by Pol is different from that of other DNA polymerases and reveals that Pol readily uses template-primers with limited base pair homology at the primer terminus. This ability, together with an ability to fill short gaps in DNA at low dNTP concentrations, is consistent with a role for mammalian Pol in non-homologous end-joining. This may include non-homologous end-joining of strand breaks resulting from DNA damage, because Pol has intrinsic 5 ,2 -deoxyribose-5-phosphate lyase activity.

Section: Fidelity Of Dna Polymerasementioning

confidence: 81%

Section: Fidelity Of Dna Polymerasementioning

confidence: 99%

The Frameshift Infidelity of Human DNA Polymerase λ

Bębenek

García‐Díaz

Blanco

et al. 2003

111

145

“…Similar procedures were used for expression and purification of a 39-kDa Cterminal fragment of pol (amino acids 242-575; details to be presented elsewhere). Hexahistidine-tagged human pol and pol were expressed in Saccharomyces cerevisiae and purified as described previously (10). Except where specifically noted, the bacterially expressed pol was used for all experiments.…”

Section: Methodsmentioning

confidence: 99%

Implication of DNA Polymerase λ in Alignment-based Gap Filling for Nonhomologous DNA End Joining in Human Nuclear Extracts

Lee¹,

Blanco

Zhou³

et al. 2004

Self Cite

204

182

Accurate repair of free radical-mediated DNA doublestrand breaks by the nonhomologous end joining pathway requires replacement of fragmented nucleotides in the aligned ends by a gap-filling DNA polymerase. Nuclear extracts of human HeLa cells, supplemented with recombinant XRCC4-DNA ligase IV complex (XRCC4/ligase IV), were capable of accurately rejoining model double-strand break substrates with a 1-or 2-base gap, and the gap-filling step was dependent on XRCC4/ligase IV. To determine what polymerase was responsible for gap filling, end joining was examined in the presence of polyclonal antibodies against each of two prime candidate enzymes, DNA polymerases and , both of which were present in the extracts. For a DNA substrate with partially complementary 3 overhangs and a 2-base gap, antibodies to polymerase completely eliminated both gap filling and accurate end joining, whereas antibodies to polymerase had little effect. Immunodepletion of polymerase , but not polymerase , likewise blocked both gap filling and end joining, and both functions could be restored by addition of recombinant polymerase . Recombinant polymerase , and a truncated polymerase lacking the Brca1 C-terminal domain, were at least 10-fold less active in restoring gap filling to the immunodepleted extracts, and polymerase ␤ was completely inactive. The results suggest that polymerase is the primary gap-filling polymerase for accurate nonhomologous end joining, and that the Brca1 C-terminal domain is required for this activity.

“…Such a misalignment incorporation mechanism facilitated by pol ␤ has already been described during TLS of an abasic site (34,35), an 8-oxodeoxyguanosine (36), and propano-deoxyguanosine lesions (37). This specific ability to catalyze template misalignment by searching microhomology sequence is shared by pol , another member of the DNA polymerase X family (38). pol ␤-dependent Extension of Primers with Two Bases Opposite the CPD or the (6-4)TT-To investigate whether pol ␤-dependent incorporated nucleotides opposite the two damaged bases could be extended, we used 18-mer primers whose termini were located directly opposite the CPD or (6-4)TT (Fig.…”

Section: Specificity Of Pol ␤-Dependent Incorporation Opposite the Cpmentioning

confidence: 80%

A Role for DNA Polymerase β in Mutagenic UV Lesion Bypass

Servant

Cazaux

Bieth

et al. 2002

We report here that DNA polymerase ␤ (pol ␤), the base excision repair polymerase, is highly expressed in human melanoma tissues, known to be associated with UV radiation exposure. To investigate the potential role of pol ␤ in UV-induced genetic instability, we analyzed the cellular and molecular effects of excess pol ␤. We firstly demonstrated that mammalian cells overexpressing pol ␤ are resistant and hypermutagenic after UV irradiation and that replicative extracts from these cells are able to catalyze complete translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). By using in vitro primer extension reactions with purified pol ␤, we showed that CPD as well as, to a lesser extent, the thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct, were bypassed. pol ␤ mostly incorporates the correct dATP opposite the 3 -terminus of both CPD and the (6-4) photoproduct but can also misinsert dCTP at a frequency of 32 and 26%, respectively. In the case of CPD, efficient and error-prone extension of the correct dATP was found. These data support a biological role of pol ␤ in UV lesion bypass and suggest that deregulated pol ␤ may enhance UV-induced genetic instability.Exposure of cells to UV light results in the formation of a variety of lesions in their DNA, the most common being cyclobutane pyrimidine dimers (CPD) 1 and pyrimidine-pyrimidone (6-4) photoproducts ((6-4)PP) at adjacent pyrimidines (1). Unrepaired, these lesions can interfere with normal DNA metabolism including DNA replication, eventually resulting in mutations that lead to carcinogenesis and/or cell death. To maintain their genetic integrity, cells have evolved multiple pathways to repair various types of DNA damage, such as nucleotide excision and base excision repair pathways (1). However, all lesions on the genome cannot be repaired efficiently by these processes in time for DNA replication, and some types of lesions are repaired very inefficiently. To prevent cell death through arrested DNA replication at unrepaired lesions, cells have a mechanism, referred to as translesion synthesis, that allows DNA synthesis to proceed past lesions and employs specialized DNA polymerases for promoting continued nascent strand extension.In human cells, recent genetic and biochemical studies suggest that translesion synthesis (TLS) past a CPD-TT or a (6-4)TT lesion could be facilitated by at least four DNA polymerases, pol , , , and . In the case of pol , this process appears to be efficient and largely accurate opposite a CPD (2), whereas it could be mutagenic and limited at the 3ЈT opposite a (6-4)TT (3). Overexpression of the antisense mRNA of Rev3, one of the components of pol , leads to a dramatic drop in the extent of UV-induced mutagenesis (4), thereby implicating human pol as having a pivotal role in error-prone translesion replication in normal cells. Indeed, pol can catalyze an efficient extension of nucleotides inserted opposite the 3ЈT of both CPD and (6-4)TT lesions (3, 5). Another DNA polymerase, pol , shows similar propertie...