A Role for DNA Polymerase β in Mutagenic UV Lesion Bypass

Abstract

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...

Structure of DNA polymerase β with a benzo[ c ]phenanthrene diol epoxide-adducted template exhibits mutagenic features

“…Although the primary cellular role of Pol ␤ is to provide lyase and nucleotidyl transferase activities during BER of simple DNA base lesions, Pol ␤ has also been implicated in the replication bypass of a variety of bulky DNA lesions. Over-expression of Pol ␤ can result in decreased sensitivity to agents that generate bulky DNA adducts, such as cisplatin (30), or UV radiation (31). In vitro assays demonstrate the ability of Pol ␤ to bypass UVinduced (31) and cisplatin-induced DNA lesions (32).…”

Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy

“…More recently, Albertella et al (2005) found that Pol b was overexpressed twofold or greater at the mRNA and protein levels in approximately one-third of all the tumors sampled compared with normal tissues, notably in 40% of colon cancer and two out of three malignant melanoma cases studied. Replicative extracts from malignant melanoma cells expressing high levels of Pol b are able to catalyse complete translesion replication of bulky adducts such as cyclobutane pyrimidine dimers (Servant et al, 2002).…”

DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

“…PolX shows amino acid sequence similarities with eukaryotic Polb (Aravind & Koonin, 1999), which has been shown to be a BER and DNA lesion bypass repair enzyme (Kubota et al, 1996;Servant et al, 2002;Dianova et al, 2004). Unlike replicative DNA polymerases such as DNA Pola, DNA Polc and DNA PolIII, which can polymerize long-stretch DNA (Holmes et al, 1990;Lahue et al, 1989), the DNA repair Polb catalyses the short patch BER function by incorporation of one or two nucleotides on damaged sites similar to very short patch repair in bacteria (4) separately, and together without DNA ligase (5-9) and with DNA ligase (10-13) in the presence of dTTP (5, 10), dCTP (6, 13), dGTP (8, 11), dATP (7, 12) and all four dNTPs (9) under standard excision repair assay conditions as described in Methods.…”

“…Apart from replicative DNA polymerases, the D. radiodurans genome was annotated for the X-family DNA-repair polymerase (PolX) encoded by DR0467, which is homologous to eukaryotic DNA polymerase b (Polb) (Aravind & Koonin, 1999). Polb plays a crucial role in BER and UV lesion bypass DNA synthesis in mammals (Kubota et al, 1996;Servant et al, 2002;Dianova et al, 2004). D. radiodurans is endowed with a strong oxidative stress-tolerance mechanism, which contributes to the extraordinary radiation tolerance of this bacterium (Markillie et al, 1999).…”