Gareth A. Cromie

Control of crossing over.

The Holliday junction is a central intermediate in homologous recombination. It consists of a four-way structure that can be resolved by cleavage to give either the crossover or noncrossover products observed. We show here that the formation of these products is controlled by the E. coli resolvasome (RuvABC) in such way that double-strand break repair (DSBR) leads to crossing over and single-strand gap repair (SSGR) does not lead to crossing over. We argue that the positioning of the RuvABC complex and its consequent direction of junction-cleavage is not random. In fact, the action of the RuvABC complex avoids crossing over in the most commonly predicted situations where Holliday junctions are encountered in DNA replication and repair. Our observations suggest that the positioning of the resolvasome may provide a general biochemical mechanism by which cells can control crossing over in recombination.

Helicobacter pylori AddAB helicase-nuclease and RecA promote recombination-related DNA repair and survival during stomach colonization

Helicobacter pylori colonization of the human stomach is characterized by profound disease‐causing inflammation. Bacterial proteins that detoxify reactive oxygen species or recognize damaged DNA adducts promote infection, suggesting that H. pylori requires DNA damage repair for successful in vivo colonization. The molecular mechanisms of repair remain unknown. We identified homologues of the AddAB class of helicase‐nuclease enzymes, related to the Escherichia coli RecBCD enzyme, which, with RecA, is required for repair of DNA breaks and homologous recombination. H. pylori mutants lacking addA or addB genes lack detectable ATP‐dependent nuclease activity, and the cloned H. pylori addAB genes restore both nuclease and helicase activities to an E. coli recBCD deletion mutant. H. pylori addAB and recA mutants have a reduced capacity for stomach colonization. These mutants are sensitive to DNA damaging agents and have reduced frequencies of apparent gene conversion between homologous genes encoding outer membrane proteins. Our results reveal requirements for double‐strand break repair and recombination during both acute and chronic phases of H. pylori stomach infection.

Recombinational repair of chromosomal DNA double-strand breaks generated by a restriction endonuclease.

DNA double‐strand break repair can be accomplished by homologous recombination when a sister chromatid or a homologous chromosome is available. However, the study of sister chromatid double‐strand break repair in prokaryotes is complicated by the difficulty in targeting a break to only one copy of two essentially identical DNA sequences. We have developed a system using the Escherichia coli chromosome and the restriction enzyme EcoKI, in which double‐strand breaks can be introduced into only one sister chromatid. We have shown that the components of the RecBCD and RecFOR ‘pathways’ are required for the recombinational repair of these breaks. Furthermore, we have shown a requirement for SbcCD, the prokaryotic homologue of Rad50/Mre11. This is the first demonstration that, like Rad50/Mre11, SbcCD is required for recombination in a wild‐type cell. Our work suggests that the SbcCD–Rad50/Mre11 family of proteins, which have two globular domains separated by a long coiled‐coil linker, is specifically required for the co‐ordination of double‐strand break repair reactions in which two DNA ends are required to recombine at one target site.

The fission yeast BLM homolog Rqh1 promotes meiotic recombination.

RecQ helicases are found in organisms as diverse as bacteria, fungi, and mammals. These proteins promote genome stability, and mutations affecting human RecQ proteins underlie premature aging and cancer predisposition syndromes, including Bloom syndrome, caused by mutations affecting the BLM protein. In this study we show that mutants lacking the Rqh1 protein of the fission yeast Schizosaccharomyces pombe, a RecQ and BLM homolog, have substantially reduced meiotic recombination, both gene conversions and crossovers. The relative proportion of gene conversions having associated crossovers is unchanged from that in wild type. In rqh1 mutants, meiotic DNA double-strand breaks are formed and disappear with wild-type frequency and kinetics, and spore viability is only moderately reduced. Genetic analyses and the wild-type frequency of both intersister and interhomolog joint molecules argue against these phenotypes being explained by an increase in intersister recombination at the expense of interhomolog recombination. We suggest that Rqh1 extends hybrid DNA and biases the recombination outcome toward crossing over. Our results contrast dramatically with those from the budding yeast ortholog, Sgs1, which has a meiotic antirecombination function that suppresses recombination events involving more than two DNA duplexes. These observations underscore the multiple recombination functions of RecQ homologs and emphasize that even conserved proteins can be adapted to play different roles in different organisms.

Activation of An Alternative, Rec12 (Spo11)-independent Pathway of Fission Yeast Meiotic Recombination in the Absence of a DNA Flap Endonuclease

Spo11 or a homologous protein appears to be essential for meiotic DNA double-strand break (DSB) formation and recombination in all organisms tested. We report here the first example of an alternative, mutationally activated pathway for meiotic recombination in the absence of Rec12, the Spo11 homolog of Schizosaccharomyces pombe. Rad2, a FEN-1 flap endonuclease homolog, is involved in processing Okazaki fragments. In its absence, meiotic recombination and proper segregation of chromosomes were restored in rec12Δ mutants to nearly wild-type levels. Although readily detectable in wild-type strains, meiosis-specific DSBs were undetectable in recombination-proficient rad2Δ rec12Δ strains. On the basis of the biochemical properties of Rad2, we propose that meiotic recombination by this alternative (Rec*) pathway can be initiated by non-DSB lesions, such as nicks and gaps, which accumulate during premeiotic DNA replication in the absence of Okazaki fragment processing. We compare the Rec* pathway to alternative pathways of homologous recombination in other organisms.

Sequence interruptions in enterobacterial repeated elements retain their ability to encode well‐folded RNA secondary structures

Recently, attention has been focused on internally deleted examples of the enterobacterial repetitive sequences BEE95 and ERIC (also known as IRU) and the mechanism by which such deletions might occur (Sharp and Leach, 1996, Mol Microbiol 22: 1055–1056). These enterobacterial repetitive sequences are imperfect palindromes (Sharples and Lloyd, 1990, Nucleic Acids Res 18: 6502– 6508; Hulton et al., 1991, Mol Microbiol 5: 825-834) and their ability to form hairpin structures, while in singlestranded form, has led to the suggestion that replication slippage, within the hairpin, could occur between directly repeated motifs resulting in the deletion events (Sharp and Leach, 1996, Mol Microbiol 22: 1055–1056). We have identified three ERIC sequences that possess extensive internal insertions rather than deletions. These insertions show some sequence similarity to ERIC itself. ERICs are capable of forming two different secondary structures in single-stranded RNA. This depends on which of the two strands of the DNA ERIC is transcribed: the one carrying the ERIC sequence as usually referenced (the canonical strand), or its complement. In the case of the consensus ERIC sequence the structure formed by the canonical strand in RNA is more stable. If RNA secondary structure is important for ERIC function, then this suggests it is the canonical strand that is active. The interrupted ERICs that were identified are all capable of forming similar secondary structures when folded in RNA, using the canonical strand. These structures also possess regions of correct ERIC secondary structure. The three interrupted ERIC sequences were identified by database search. The program MPSRCH_NNA from the MPSRCH suite (J. F. Collins; Release 3.0, Oxford Molecular Ltd) was used to search the EMBL prokaryotic database using an ERIC consensus sequence as the query. This program is a fast implementation of the Smith–Waterman algorithm (Smith and Waterman, 1981, J Mol Biol 147: 195–197), with the affine gap-extension algorithm of Gotoh (Gotoh, 1990, Bull Math Biol 52: 359–373). The parameters were set to a mismatch penalty of one, a gapopen penalty of 12 and a gap-extend penalty of zero. This enabled the identification of several internally deleted ERIC sequences, but also three examples where an intact ERIC has been interrupted by an inserted sequence of the 69–75 base range (Fig. 1A). The three ERICs of interest lie 38 to the pddC gene of Klebsiella oxytoca (E1), between the nirD and nirC genes of Salmonella typhimurium (E2), and between the topA and cysB genes of S. typhimurium (E3). E1 contains an insertion of 69 bp, E2 one of 70 bp and E3 an insertion of 75 bp. As well as being of comparable lengths, these sequences occur at similar positions with respect to the ends of the ERIC sequences. The insertion has occurred between ERIC bases 45 and 46 of E1 and bases 42 and 43 of E2, counting from the 58 end of the canonical ERIC sequence (Fig. 1A). In E3 the insertion lies in the region between ERIC bases 86 and 88 (base 87 does not match the E3 sequence at either end of the insert, so the insertion site cannot be precisely defined). However, this corresponds to an insertion in the region between bases 40 and 42 counting from the 38 end. Therefore an insertion has occurred approx. 40 bp from the 58 end of the ERICs in two cases and approx. 40 bases from the 38 end in another case. If the interrupting sequences are compared to one another, they share large regions of homology (Fig. 1, A and 1B). Interestingly, these sequences are present on the same strand, relative to the 58 end of the ERIC, in E3 as well as E1 and E2, even though the E3 event has occurred on the ‘other side’ of the ERIC. Comparison of the inserted sequences and the ERIC consensus sequences also shows a certain degree of similarity, suggesting that the two may be related (Fig. 1C). The similarity of the position and sequence of the insertions suggests a common mechanism of formation. It seems unlikely that E1 and E2 could be related by descent to E3, with its different insertion site. The predicted ability of ERICs to form secondary structure in single-stranded RNA has been remarked upon from the time of their discovery and several suggestions about possible functions for these structures have been proposed (Sharples and Lloyd, 1990, ibid.). However, any ERIC is potentially capable of forming two different RNAs depending on whether the strand carrying the canonical sequence or its complement is transcribed, and these structures are not equivalent. The ERIC canonical sequence and its complement were folded using Michael Zuker’s RNAfold server at http::/ /www.ibc.wustl.edu/,zuker/.cgi. The canonical sequence formed a considerably more stable structure with a free energy of 153.8 kcal mol compared to a free energy of 141.3 kcal mol for the complementary sequence. If formation of stable secondary structures in RNA is important for ERIC function this suggests that it could be the canonical strand that mediates ERIC activity. Recently, a new motif of RNA secondary Molecular Microbiology (1997) 24(6), 1311–1315