Journal Article

Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant

Sharon K. Bullock, William K. Kaufmann and Marila Cordeiro-Stone

in Carcinogenesis

Volume 22, issue 2, pages 233-241
Published in print February 2001 | ISSN: 0143-3334
Published online February 2001 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/22.2.233
Enhanced S phase delay and inhibition of replication of an undamaged shuttle vector in UVC-irradiated xeroderma pigmentosum variant

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Xeroderma pigmentosum variant (XP-V) cells are defective in bypass replication of UVC-induced thymine dimers in DNA because they lack a novel DNA polymerase (polymerase η). In this study the effects of UVC on S phase cells were compared in fibroblasts derived from normal donors (IDH4) and XP-V patients (CTag) and immortalized by expression of the SV40 large T antigen. These transformed fibroblasts did not activate the G1 checkpoint or inhibit replicon initiation when damaged by UVC or γ-rays. The transformed XP-V cells (CTag) retained the increased sensitivity to UVC-induced inhibition of DNA strand growth previously observed with their diploid counterpart. Cell cycle progression analyses showed that CTag cells displayed a stronger S phase delay than transformed fibroblasts from normal individuals (IDH4) after treatment with only 2 J/m2 UVC. Low doses of UVC also caused a lag in CTag cell proliferation. The extent of replication of an episomal DNA (pSV011), not previously exposed to radiation, was measured after the host cells were irradiated with 1–3 J/m2 UVC. Replication of pSV011 was barely affected in irradiated IDH4 cells. Plasmid replication was inhibited by 50% in irradiated CTag cells and this inhibition could not be accounted for by increased killing of host cells by UVC. These results suggest that even in transformed cells UVC induces DNA damage responses that are reflected in transient cell cycle arrest, delay in proliferation and inhibition of episomal DNA replication. These responses are enhanced in CTag cells, presumably because of their bypass replication defect. The accumulation of replication complexes blocked at thymine dimers and extended single-stranded regions in chromosomal DNA might sequester replication factors that are needed for plasmid and chromosomal replication. Alternatively, aberrant replication structures might activate a signal transduction pathway that down-regulates DNA synthesis.

Keywords: ATM, gene mutated in ataxia telangectasia; BrdU, bromodeoxyuridine; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; MEM, minimum essential medium; PI, PhosphorImager units; pol η, DNA polymerase η; PRR, post-replication repair; RPA, replication protein A (single-stranded DNA-binding protein); XP-V, xeroderma pigmentosum variant.

Journal Article.  8332 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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