Introduction Targeted nucleotide exchange, the underlying mechanism of gene repair, can direct the alteration of single bases in biological systems including cell-free extracts, Escherichia coli, Saccharomyces cerevisiae, mammalian cells in culture and a range of animal models including mouse, rat and dog (Alexeev et al., 2000; Alexeev and Yoon, 1998; Bandyopadhyay et al., 1999; Bartlett et al., 2000; Brachman and Kmiec, 2002; Brachman and Kmiec, 2003; Campbell et al., 1989; Cole-Strauss et al., 1996; Cole-Strauss et al., 1999; Ellis et al., 2001; Gamper et al., 2000; Kmiec, 1999; Kren et al., 1997; Kren et al., 1999; Liu et al., 2001; Moerschell et al., 1988; Parekh-Olmedo et al., 2001; Rando et al., 2000; Rice et al., 2001; Santana et al., 1998; Vasquez et al., 2001; Yamamoto et al., 1992a; Yamamoto et al., 1992b; Yoon et al., 1996). Now, efforts are under way to elucidate the mechanism of this dynamic process so that correction efficiencies can be stabilized and eventually raised to therapeutically useful levels. Genetic studies in yeast have revealed that DNA repair proteins play regulatory roles in oligonucleotide-directed repair (Brachman and Kmiec, 2003; Liu et al., 2002a). Additionally, transcriptional activity of the target gene has been found to provide a more open chromatin conformation, which enables a higher frequency of gene repair (Igoucheva et al., 2003; Liu et al., 2002b). A strand bias has repeatedly been observed in which the untranscribed strand is corrected more efficiently (Igoucheva et al., 2001; Igoucheva et al., 2003; Liu et al., 2001; Liu et al., 2002b; Nickerson and Colledge, 2003) (Y. Hu, L. Liu, L. Ferrara and E.B.K., unpublished) and lends further support for a productive role of transcription in the gene repair process. This transcriptional strand bias is probably due to the availability of the untranscribed strand for oligonucleotide alignment, in contrast to the transcribed strand, which is more encumbered by the transcriptional machinery. Furthermore, experiments conducted in vitro indicate that RNA polymerase is able to displace the oligonucleotide from the transcribed strand, reducing its potential for directing a gene repair event (Liu et al., 2002b). Transcriptional activity alone, however, does not always dictate the strand bias of correction. For example, when targeted nucleotide exchange takes place on the S. cerevisiae CYC1 gene, a gene involved in cytochrome c metabolism, a repair bias is observed that favours the transcribed strand (Brachman and Kmiec, 2003; Yamamoto et al., 1992b). Similar results have now been reported in two unrelated mammalian systems (Bertoni et al., 2003; Bertoni and Rando, 2003; Sorensen et al., 2003), suggesting that processes other than transcription can impact strand bias. In the current work, we examine the possibility that DNA replication influences the process of gene repair strand preference. Support for this notion comes from important studies in prokaryotes in which DNA replication has already been shown to dominate the strand bias phenomenon (Ellis et al., 2001; Zhang et al., 2003). Recombination within the gal operon of E. coli is higher when oligonucleotides complementary to the lagging strand of replication are used. galK targeting revealed that the bias for the lagging strand remained intact even when the gal operon was inverted, reversing the strands of replication 3867 The repair of point mutations can be directed by modified single-stranded DNA oligonucleotides and regulated by cellular activities including homologous recombination, mismatch repair and transcription. Now, we report that DNA replication modulates the gene repair process by influencing the frequency with which either DNA strand is corrected. An SV40-virus-based system was used to investigate the role of DNA synthesis on gene repair in COS-1 cells. We confirm that transcription exerts a strand bias on the gene repair process even when correction takes place on actively replicating templates. We were able to distinguish between the influences of transcription and replication on strand bias by changing the orientation of a gene encoding enhanced green fluorescent protein relative to the origin of replication, and confirmed the previously observed bias towards the untranscribed strand. We report that DNA replication can increase the level of untranscribed strand preference only if that strand also serves as the lagging strand in DNA synthesis. Furthermore, the effect of replication on gene repair frequency and strand bias appears to be independent of certain mismatched base pairs and oligonucleotide length. Key words: DNA repair, SV40, COS cells Summary DNA replication and transcription direct a DNA strand bias in the process of targeted gene repair in mammalian cells Erin E. Brachman and Eric B. Kmiec* Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA *Author for correspondence (e-mail: ekmiec@udel.edu) Accepted 6 April 2004 Journal of Cell Science 117, 3867-3874 Published by The Company of Biologists 2004 doi:10.1242/jcs.01250 Research Article