LETTERS Break-induced replication and telomerase- independent telomere maintenance require Pol32 John R. Lydeard 1 , Suvi Jain 1 , Miyuki Yamaguchi 1 & James E. Haber 1 Break-induced replication (BIR) is an efficient homologous recombination process to initiate DNA replication when only one end of a chromosome double-strand break shares homology with a template 1–5 . BIR is thought to re-establish replication at stalled and broken replication forks and to act at eroding telo- meres in cells that lack telomerase in pathways known as ‘alterna- tive lengthening of telomeres’ (reviewed in refs 2, 6). Here we show that, in haploid budding yeast, Rad51-dependent BIR induced by HO endonuclease requires the lagging strand DNA Pola-primase complex as well as Pold to initiate new DNA synthesis. Pole is not required for the initial primer extension step of BIR but is required to complete 30 kb of new DNA synthesis. Initiation of BIR also requires the nonessential DNA Pold subunit Pol32 primarily through its interaction with another Pold subunit, Pol31. HO- induced gene conversion, in which both ends of a double-strand break engage in homologous recombination, does not require Pol32. Pol32 is also required for the recovery of both Rad51- dependent and Rad51-independent survivors in yeast strains lacking telomerase. These results strongly suggest that both types of telomere maintenance pathways occur by recombination- dependent DNA replication. Thus Pol32, dispensable for replica- tion and for gene conversion, is uniquely required for BIR; this finding provides an opening into understanding how DNA rep- lication re-start mechanisms operate in eukaryotes. We also note that Pol32 homologues have been identified both in fission yeast and in metazoans where telomerase-independent survivors with alternative telomere maintenance have also been identified 2,6,7 . When a double-strand break (DSB) occurs in the middle of a chromosome, it can be efficiently repaired by gene conversion, using homologous sequences on a sister chromatid, a homologous chro- mosome, or at an ectopic location 1,8,9 . However, if only one end of a DSB shares homology with other sequences in the genome, cells rely on recombination-dependent DNA replication (known as break- induced replication, BIR) to restore genome integrity 2–5 (see also Supplementary Fig. 1). Such events are believed to occur at stalled replication forks, where endonuclease cleavage produces one par- tially replicated chromatid and an intact chromatid 10,11 . BIR can re- establish a unidirectional replication fork, which can proceed to the end of the template chromatid or until it meets a converging replica- tion fork. BIR also could explain how rare survivors arise in Saccharomyces cerevisiae cells lacking telomerase, in which chromosome ends become eroded and recombinogenic 12 . Virtually all telomerase-deficient sur- vivors require the key homologous recombination protein Rad52, but there are two distinct survivor pathways that differ in their genetic and substrate requirements 13,14 . Type I Rad51-dependent survivors result from recombination between subtelomeric repeat sequences, whereas Type II Rad51-independent—but Rad50- and Rad59-dependent— survivors exhibit recombination between imperfect nucleotide TG 1-3 sequences of the telomeres themselves. It is believed that these events must involve DNA replication—possibly rolling circle replica- tion of an excised circular template 2 —but there has not yet been any direct demonstration of the involvement of leading- and lagging- strand DNA polymerases in these repair events. We developed a haploid system to study BIR in budding yeast, where a galactose-inducible HO endonuclease creates a DSB adjacent to the 59 part of the CAN1 gene (designated CA) in a nonessential terminal region of chromosome V-left (Chr. V-L). The DSB shares homology, only on the centromere-proximal side, with a 39 segment of the CAN1 gene (called AN1) that is inserted on another chro- mosome (Fig. 1a). Successful recombination results in a non- reciprocal translocation, restoring an intact CAN1 gene that renders the cells canavanine sensitive, but leads to the loss of the hygromycin- resistance HPH-MX marker distal to the DSB. Interchromosomal Rad51- and Rad52-dependent recombination yields BIR products, the frequency of which increases from 21% to 31% as the length of shared homology on Chr. XI increases from 1,157 to 2,977 base pairs (bp) (Fig. 1b and Supplementary Table 1). BIR was more efficient in 1 MS029 Rosenstiel Centre, Brandeis University, Waltham, Massachusetts 02454-9110, USA. a A A A A A A HO Probe Probe Probe AN1 LEU2 LEU2 LEU2 P2 P2 P2 P1 CAN1 Chr. XI A A CA HPH P1 Probe Chr. V Chr. XI Chr. V AN1 0 1 3 6 8 10 12 24 Time (h) Southern blot PCR Product (percentage of maximum repair) 70 60 50 40 30 20 10 80 0 b 01368 S Chr. XI BIR Cut Chr. V 101224 Time (h) Figure 1 | Experimental system to study BIR and kinetics of DSB repair. a, An HPH-MX marked HO endonuclease cut site (grey bars) integrated into Chr. V-L deletes the 39 end of CAN1. The LEU2-marked AN1 donor sharing 1,157 bp of homology with CAN is integrated into Chr. XI. b, PCR with primers P1 and P2 monitors initiation of new DNA synthesis. Southern blot analysis of AvaI-digested DNA (marked by A) monitors completion of BIR. Lane S contains DNA from a colony in which BIR occurred. Kinetics of repair is shown for PCR and Southern blot assays of BIR induced in cycling cells. Error bars represent s.d. Vol 448 | 16 August 2007 | doi:10.1038/nature06047 820 Nature ©2007 Publishing Group