Published online January 12, 2005 280–288 Nucleic Acids Research, 2005, Vol. 33, No. 1 doi:10.1093/nar/gki168 The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase b defective mammalian cells Barbara Pascucci1,2, Maria Teresa Russo 2 , Marco Crescenzi2, Margherita Bignami2 and Eugenia Dogliotti2,* 1Istituto di Cristallografia, CNR, Sezione di Roma, PO Box 10, 00016 Monterotondo Stazione, Roma, Italy and 2Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Roma, Italy Received September 10, 2004; Revised November 26, 2004; Accepted December 15, 2004 ABSTRACT DNA polymerase (Pol) b mouse embryonic fibro- blasts provide a useful cell system to investigate the effects of alterations in base excision repair (BER) on genome stability. These cells are character- ized by hypersensitivity to the cytotoxic effects of methyl methanesulfonate (MMS) and by decreased repair of the MMS-induced DNA single strand breaks (SSB). Here, we show that, in the absence of Pol b, SSB accumulate in G1 phase cells, accompanied by the formation of proliferating cell nuclear antigen foci in the nuclei. When replicating Pol b cells are treated with MMS, a rapid phosphorylation of histone H2AX is detected in the nuclei of S phase cells, indicating that double strand breaks (DSB) are formed in response to unrepaired SSB. This is followed by relocalization within the nuclei of Rad51 protein, which is essential for homologous recombination (HR). These findings are compatible with a model where, in mammalian cells, unrepaired SSB produced during BER are sub- strates for the HR pathway via DSB formation. This is an example of a coordinated effort of two different repair pathways, BER and HR, to protect mammalian cells from alkylation-induced cytotoxicity. INTRODUCTION Single strand breaks (SSB) can arise directly or indirectly as normal intermediates of base excision repair (BER). They are induced by both exogenous and endogenous processes, thus posing a continuous threat to genetic integrity. In higher eukar- yotes, it is known that SSB repair occurs via two alternative pathways: a DNA polymerase (Pol) b-dependent pathway (short patch BER) and a proliferating cell nuclear antigen (PCNA)-dependent pathway (long patch BER) [for a review see (1)]. SSB repair is a highly coordinated process and a defect in this ordered chain of enzymatic steps is hampered in vivo as testiﬁed by the dramatic phenotype of Pol b knocked out mice. Transgenic mice with a homozygous mutation in Pol b gene are non-viable after birth (2). Pol b mouse defective cells are hypersensitive to MMS and are less efﬁcient in the rejoining of induced SSB (3–6). Pol b has a dual role in the indirect SSB formed during BER: Pol b inserts a single nucleotide at the gap site and repairs the 50-deoxyribose phosphate terminus (50-dRP) created by AP endonuclease-1 (APE1). It has been elegantly shown that it is the loss of the 50-dRP removal by the 50-dRP lyase activity of Pol b that renders mouse ﬁbroblasts alkylation- hypersensitive (7). Moreover, Pol b mutant mouse cells exhibit increased frequencies of sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) after methyl methanesul- fonate (MMS) treatment (4,8). If SSB are not properly repaired, they may result in double strand breaks (DSB) in replicating DNA. H2AX phosphoryla- tion (gH2AX) has been found at the sites of DSB in chromo- somal DNA (9). Because gH2AX appears within minutes after ionizing radiation (IR), gH2AX focus formation is considered to be a sensitive and selective signal for the existence of DSB (10). DSB may be repaired either by direct end-joining of the broken ends (non homologous end joining, NHEJ) or by homo- logous recombination (HR). Although mammalian cells are presumed to repair DSB predominantly by NHEJ (11,12), accumulating experimental evidence suggests that HR also plays an essential role in the mammalian DSB repair (13–15). One of the central components of HR is Rad51 protein that forms nucleoprotein ﬁlament on single-stranded DNA, medi- ating homologous pairing and strand-exchange reactions between single-stranded DNA and homologous double- stranded DNA (16–19). After DNA damage, Rad51 protein is detected in multiple discrete foci in the nucleoplasm. *To whom correspondence should be addressed. Tel: +39 06 49902580; Fax: +39 06 49903650; Email: dogliott@iss.it The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use permissions, please contact journals.permissions@oupjournals.org. ª 2005, the authors Nucleic Acids Research, Vol. 33 No. 1 ª Oxford University Press 2005; all rights reserved