Bleomycin-induced DNA repair synthesis in permeable human fibroblasts: mediation of long-patch and short-patch repair by distinct DNA polymerases

Abstract

Treatment of permeable human fibroblasts with bleomycin elicits DNA repair synthesis that is only partially sensitive to aphidicolin, an inhibitor of mammalian DNA polymerases alpha and delta. Inhibition of long-patch repair synthesis by omission of the three unlabeled deoxyribonucleoside triphosphates (dNTPs) selectively eliminates the aphidicolin-sensitive component. The majority of this residual aphidicolin-resistant repair synthesis is contained in ligated patches as revealed by resistance to exonuclease III. Determination of repair patch length by bromodeoxyuridine-induced density shift under conditions where essentially all of the repair synthesis is sensitive or resistant to aphidicolin yielded values of approximately 20 and 4 nucleotides per patch, respectively. On the basis of these data and the relative sensitivity of bleomycin-induced repair synthesis to N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP), 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), and N-ethylmaleimide (NEM), long-patch repair is attributed to DNA polymerase delta and short-patch repair to DNA polymerase beta.

Defective DNA Ligation during Short-Patch Single-Strand Break Repair in Ataxia Oculomotor Apraxia 1

“…This process would not be detected by the shortpatch repair assays employed in the current work, because oligonucleotide duplexes of the type employed here are not good substrates for long-patch repair reactions and because cleavage of the single-strand flap would remove the 32 P label in our substrates. However, to determine whether long-patch repair might compensate for the loss of short-patch repair in aprataxin-defective cells, we exploited the observation that long-patch SSBR employs the aphidicolin-sensitive DNA polymerases Pol ␦ and/or Pol ε (10,13,22). Because inhibition of Pol ␦ and/or Pol ε also prevents DNA replication during S phase and can thus impact DNA metabolism independently of long-patch SSBR, we employed quiescent primary astrocytes for these experiments.…”

“…While our in vitro assay was not capable of measuring long-patch repair reactions, this process might operate efficiently at adenylated nicks within a chromosomal context. To examine this possibility, we compared chromosomal SSBR rates in WT and Aptx Ϫ/Ϫ quiescent mouse neural astrocytes in the presence and absence of aphidicolin, an inhibitor of the DNA polymerases Pol ␦ and Pol ε, which are implicated in long-patch repair reactions (10,13,22). Strikingly, whereas aphidicolin did not measurably reduce SSBR rates in WT astrocytes following H 2 O 2 treatment, it significantly slowed SSBR in Aptx Ϫ/Ϫ astrocytes (Fig.…”

Disruption of theAtREV3Gene Causes Hypersensitivity to Ultraviolet B Light and γ-Rays in Arabidopsis: Implication of the Presence of a Translesion Synthesis Mechanism in Plants [W]

“…Incorporation of BrdU often is used as an index of DNA replication and cell proliferation (Gratzner, 1982;Nagata et al, 2000). Although BrdU is known to be incorporated in DNA during dark repair and TLS (DiGiuseppe and Dresler, 1989;Srivastava et al, 1993), the amount that is incorporated by these processes is quite small compared with the amount incorporated during replication. Without UV-B light treatment, the tissues of wild-type and rev3-1 roots were well stained with the anti-BrdU antibody ( Figures 7A and 7E), indicating that the vigorously proliferating root tip cells incorporated BrdU.…”

DNA polymerase beta null mouse embryonic fibroblasts harbor a homozygous null mutation in DNA polymerase iota

“…Although it was subsequently shown that pol α can also carry-out gap-filling DNA synthesis in a base excision repair (BER) reaction [13], the evidence continued to mount in support of pol β acting as 'the' DNA repair polymerase in the nucleus, while pol γ participated in replication and repair in mitochondria [1]. Studies continued to identify a role for pol β in the repair of damage induced by many different DNA damaging agents, including bleomycin [14,15] [21][22][23][24]. Several groups reported complete BER in vitro with pol β and additional purified proteins [22,23,25].…”