Mutagenic and Nonmutagenic Bypass of DNA Lesions byDrosophila DNA Polymerases dpolη and dpolι

Abstract

cDNA sequences were identified and isolated that encode Drosophila homologues of human Rad30A and Rad30B called drad30A and drad30B. Here we show that the C-terminal-truncated forms of the drad30A and drad30B gene products, designated dpol⌬C and dpol⌬C, respectively, exhibit DNA polymerase activity. dpol⌬C and dpol⌬C efficiently bypass a cis-syn-cyclobutane thymine-thymine (TT) dimer in a mostly error-free manner. dpol⌬C shows limited ability to bypass a 6 -4-photoproduct ((6 -4)PP) at thymine-thymine (TT-(6 -4)PP) or at thymine-cytosine (TC-(6 -4)PP) in an error-prone manner. dpol⌬C scarcely bypasses these lesions. Thus, the fidelity of translesion synthesis depends on the identity of the lesion and on the polymerase. The human XPV gene product, hpol, bypasses cis-syn-cyclobutane thymine-thymine dimer efficiently in a mostly error-free manner but does not bypass TT-(6 -4)PP, whereas Escherichia coli DNA polymerase V (UmuD 2 C complex) bypasses both lesions, especially TT-(6 -4)PP, in an error-prone manner (Tang, M., Pham, P., Shen, X., Taylor, J. S., O'Donnell, M., Woodgate, R., and Goodman, M. F. (2000) Nature 404, 1014 -1018). Both dpol⌬C and DNA polymerase V preferentially incorporate GA opposite TT-(6 -4)PP. The chemical structure of the lesions and the similarity in the nucleotides incorporated suggest that structural information in the altered bases contribute to nucleotide selection during incorporation opposite these lesions by these polymerases.DNA is frequently damaged by environmental and endogenous genotoxic agents. Although various mechanisms exist to ensure that the majority of DNA damage is recognized and repaired and the integrity of the DNA is faithfully restored, some DNA lesions escape repair and persist in the cell. Unrepaired DNA lesions can block the progress of the replication machinery. To help resolve this problem, cells have specialized polymerases that carry out translesion DNA synthesis (TLS) 1 , which is a mechanism that permits nucleotides to be incorporated opposite lesions. After TLS bypasses the DNA damage, replication can continue with the normal replication machinery downstream of the site of the damage.Recently, a new family of DNA polymerases was identified called the UmuC/DinB/Rev1/Rad30 superfamily (1-3). Several of these polymerases participate in TLS (4 -11), and members of this protein family exhibit lower fidelity and processivity than replicative DNA polymerases (10, 12-15). The lower fidelity enables these polymerases to carry out TLS and the lower processivity facilitates dissociation of these enzymes after bypass of a lesion; this is important because it allows the normal replication machinery, with high fidelity and processivity, to preferentially carry out DNA synthesis distal to the lesion. However, TLS is mutagenic when the base inserted opposite the DNA lesion is different than the base normally inserted opposite an undamaged base at that site. The chemical structure of the lesion is an important determinant for mutagenic or nonmutagenic bypass during TLS. Irr...

Pol ι Is a Candidate for the Mouse Pulmonary Adenoma Resistance 2 Locus, a Major Modifier of Chemically Induced Lung Neoplasia

“…For ex- ample, the motif SRGVLSFF (in which the conserved residues are indicated in bold) is present in residues 540 -547 of human POL and may contribute to PCNA binding (34). Another study has shown that the localization of POL protein in the nucleus requires sequences within amino acids 219 -451, and sequences within amino acids 492-539 and 636 -715 are critical for interaction between POL and POL proteins (35,36). The results in Fig.…”

TRIP/NOPO E3 ubiquitin ligase promotes ubiquitylation of DNA polymerase η

“…Polη is known to specifically bypass UV-induced lesions in yeast and mammals, and dPolη has been shown to synthesize efficiently past cyclobutane pyrimidine dimers in vitro (Ishikawa et al, 2001;Johnson et al, 1999;McCulloch et al, 2004;McDonald et al, 1997). We therefore tested the sensitivity of embryos from Exc2.15 -derived embryos (Fig.…”

“…dPolη and dPolι are functional translesion polymerases in vitro (Ishikawa et al, 2001). dPolη larvae exhibit increased sensitivity to UV irradiation, and homologous recombination is impaired in dPolη flies (Kane et al, 2012).…”

“…Three members of the Y-family of DNA polymerases are conserved in Drosophila: dPolη, dPolι and dRev1 (Ishikawa et al, 2001). dPolη and dPolι are functional translesion polymerases in vitro (Ishikawa et al, 2001).…”

Comparative analysis of in vivo interactions between Rev1 protein and other Y-family DNA polymerases in animals and yeasts

“…Drosophila constructs-Rev1 was amplified by RT-PCR of total RNA prepared by Trizol extraction of Kc cells and cloned into pACT2. Polη and Polι were amplified from pGEXdPolι and pGEX-dPolι [20] and cloned into pGBKT7. Rev7 was amplified by RT-PCR and cloned into pGBKT7.…”