Mre11/Rad50 complexes in all organisms function in the repair of DNA double-strand breaks. In budding yeast, genetic evidence suggests that the Sae2 protein is essential for the processing of hairpin DNA intermediates and meiotic double-strand breaks by Mre11/Rad50 complexes, but the biochemical basis of this functional relationship is not known. Here we demonstrate that recombinant Sae2 binds DNA and exhibits endonuclease activity on single-stranded DNA independently of Mre11/Rad50 complexes, but hairpin DNA structures are cleaved cooperatively in the presence of Mre11/Rad50 or Mre11/Rad50/Xrs2. Hairpin structures are not processed at the tip by Sae2 but rather at single-stranded DNA regions adjacent to the hairpin. Truncation and missense mutants of Sae2 inactivate this endonuclease activity in vitro and fail to complement Deltasae2 strains in vivo for meiosis and recombination involving hairpin intermediates, suggesting that the catalytic activities of Sae2 are important for its biological functions.

Cells have high-fidelity polymerases whose task is to accurately replicate the genome, and low-fidelity polymerases with specialized functions. Although some of these low-fidelity polymerases are exceptional in their ability to replicate damaged DNA and restore the undamaged sequence, they are error prone on undamaged DNA. In fact, these error-prone polymerases are sometimes used in circumstances where the capacity to make errors has a selective advantage. The mutagenic potential of the error-prone polymerases requires that their expression, activity, and access to undamaged DNA templates be regulated. Here we review these specialized polymerases with an emphasis on their biological roles.

Selective gene amplification is associated with normal development, neoplasia, and drug resistance. One class of amplification events results in large arrays of inverted repeats that are often complex in structure, thus providing little information about their genesis. We made a recombination substrate in Saccharomyces cerevisiae that frequently generates palindromic duplications to repair a site-specific double-strand break in strains deleted for the SAE2 gene. The resulting palindromes are stable in sae2⌬ cells, but unstable in wild-type cells. We previously proposed that the palindromes are formed by invasion and break-induced replication, followed by an unknown end joining mechanism. Here we demonstrate that palindrome formation can occur in the absence of RAD50, YKU70, and LIG4, indicating that palindrome formation defines a new class of nonhomologous end joining events. Sequence data from 24 independent palindromic duplication junctions suggest that the duplication mechanism utilizes extremely short (4-6 bp), closely spaced (2-9 bp), inverted repeats to prime DNA synthesis via an intramolecular foldback of a 3 end. In view of our data, we present a foldback priming model for how a single copy sequence is duplicated to generate a palindrome.[Keywords: DNA palindrome; gene amplification; MR complex; SAE2; Saccharomyces cerevisiae; BFB] Supplemental material is available at http://www.genesdev.org.

At least one of the procyclic acidic repetitive protein (PARP or

We demonstrate that the bacteriophage lambda Red functions efficiently recombine linear DNA or single-strand oligonucleotides (ss-oligos) into bacteriophage lambda to create specific changes in the viral genome. Point mutations, deletions, and gene replacements have been created. While recombineering with oligonucleotides, we encountered other mutations accompanying the desired point mutational change. DNA sequence analysis suggests that these unwanted mutations are mainly frameshift deletions introduced during oligonucleotide synthesis.

Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960′s Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called “the meiotic effect.” Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 –fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

On the basis of earlier studies with both detergent-disrupted virions (the endogenous reaction) and an in vitro reconstructed reaction, the RNase H activity associated with Moloney murine leukemia virus reverse transcriptase has been implicated in the generation of plus-strand RNA primers during reverse transcription. Here we used an oligonucleotide extension assay to show that the RNA primers remaining bound to the plus DNA strands initiated at the normal origin in the in vitro reaction are heterogeneous in length. This result indicates that, although a precise cleavage generates the 3' end of the priming RNA, RNase H exhibits less specificity at other break sites. During the endogenous reaction, a kinetic analysis of the synthesis of plus strands corresponding to different regions of the genome suggested that additional sites for the initiation of plus-strand DNA existed upstream of the normal origin. Direct analysis of fragments produced in the endogenous reaction, as well as in the in vitro reaction, confirmed the existence of upstream plus-strand initiation sites. Several of these sites were mapped to the nucleotide level by the oligonucleotide extension method. A comparison of the nucleotide sequences surrounding the upstream initiation sites with the sequence at the normal plus-strand origin revealed common features, which suggests a mechanism for plus-strand priming based on sequence recognition by the RNase H/reverse transcriptase protein. Although primer removal by RNase H is highly efficient for DNA fragments initiated at the normal origin, the RNA primers were inefficiently removed from the fragments initiated at the upstream sites. This result suggests that primer removal, like primer generation, involves sequence recognition by the enzyme.

Escherichia coli integration host factor (IHF), a DNA-binding protein, positively regulates expression of the A, clI gene. Purified IHF stimulates cII protein synthesis in vitro, suggesting a direct role for host factor in cII expression. Further evidence for a direct role for IHF was obtained with operon and gene fusions between clI and lacZ or cII and galE. Analysis of these fusions ir vivo demonstrated that IHF is essential for the initiation of cIT translation. Replacement of the entire clI coding sequence with lacZ yielded a gene fusion which was still IHF dependent. However, a cIT-galE fusion carrying a hybrid ribosome binding region expressed galE in IHF mutants. These results indicate that sequences which make cll translation IHF dependent lie between the ribosome binding region and the initiating codon of cII. Failure to translate clI activates a transcription terminator located within cIT and results in polar effects on downstream transcription. This polarity is suppressed by the A N antitermination function. When cloned into another contest, the terminator is active in both wild-type and IHF mutant strains. The amino terminus of clI is located near an IHF binding site in a region with considerable dyad symmetry. The role of IHF in cII translation may be to prevent formation of an RNA-RNA duplex that sequesters the ribosome binding site of cII. The binding of IHF might influence RNA structure by altering the rate of the dissociation of RNA from the DNA template.