Single-molecule dilution and multiple displacement amplification for molecular haplotyping

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.     

DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways.

The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.     

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.     

Novel PMS1 alleles preferentially affect the repair of primer strand loops during DNA replication

Null mutations in DNA mismatch repair (MMR) genes elevate both base substitutions and insertions/deletions in simple sequence repeats. Data suggest that during replication of simple repeat sequences, polymerase slippage can generate single-strand loops on either the primer or template strand that are subsequently processed by the MMR machinery to prevent insertions and deletions, respectively. In the budding yeast Saccharomyces cerevisiae and mammalian cells, MMR appears to be more efficient at repairing mispairs comprised of loops on the template strand compared to loops on the primer strand. We identified two novel yeast pms1 alleles, pms1-G882E and pms1-H888R, which confer a strong defect in the repair of "primer strand" loops, while maintaining efficient repair of "template strand" loops. Furthermore, these alleles appear to affect equally the repair of 1-nucleotide primer strand loops during both leading- and lagging-strand replication. Interestingly, both pms1 mutants are proficient in the repair of 1-nucleotide loop mispairs in heteroduplex DNA generated during meiotic recombination. Our results suggest that the inherent inefficiency of primer strand loop repair is not simply a mismatch recognition problem but also involves Pms1 and other proteins that are presumed to function downstream of mismatch recognition, such as Mlh1. In addition, the findings reinforce the current view that during mutation avoidance, MMR is associated with the replication apparatus.     

PCR- and ligation-mediated synthesis of marker cassettes with long flanking homology regions for gene disruption in Saccharomyces cerevisiae.

We developed a novel method for synthesizing marker-disrupted alleles of yeast genes. The first step is PCR amplification of two sequences located upstream and downstream of the reading frame to be disrupted. Due to the addition of non-specific single A overhangs by Taq DNA polymerase, each PCR product can be ligated with a marker DNA which has T residues at its 3' ends. After amplification of individual ligation products through the second PCR, both products are mixed and annealed, and the single strand is converted to a double strand by an extension reaction. The final step is PCR amplification of the fragment composed of a selectable marker and two flanking sequences with the outermost primers. This method is rapid and needs only short oligonucleotides as primers.     

Mapping the double-strand breaks at the mating-type locus in fission yeast by genomic sequencing.

In fission yeast mating-type switching is initiated by the formation of a double-strand DNA break at the mating-type locus. A prerequisite for generation of the break is some 'imprinting' of the DNA in the previous cell cycle. We have used the technique of genomic sequencing to map the position of the break directly on chromosomal DNA cleaved in vivo. On one strand the break is situated very close to the right-hand border of the expressed mat1 cassette. Cells of opposite mating type, P and M, have their breaks at slightly different positions on this strand. On the other DNA strand of both alleles the ends are probably masked by tightly bound proteins and therefore the precise nature of the break could not be determined. Since the break is stable throughout the cell cycle, these proteins may function in vivo to confer structural stability on the chromosomes having the break. The implications of these findings for models of mating-type switching are discussed.     

Deletions in Plasmid Pbr322: Replication Slippage Involving Leading and Lagging Strands

We test here whether a class of deletions likely to result from errors during DNA replication arise preferentially during synthesis of either the leading or the lagging DNA strand. Deletions were obtained by reversion of particular insertion mutant alleles of the pBR322 amp gene. The alleles contain insertions of palindromic DNAs bracketed by 9-bp direct repeats of amp sequence; in addition, bp 2 to 5 in one arm of the palindrome form a direct repeat with 4 bp of adjoining amp sequence. Prior work had shown that reversion to Ampr results from deletions with endpoints in the 8- or 4-bp repeat, and that the 4-bp repeats are used preferentially because one of them is in the palindrome. To test the role of leading and lagging strand synthesis in deletion formation, we reversed the direction of replication of the amp gene by inverting the pBR322 replication origin, and also constructed new mutant alleles with a 4-bp repeat starting counterclockwise rather than clockwise of the insertion. In both cases the 4-bp repeats were used preferentially as deletion endpoints. A model is presented in which deletions arise during elongation of the strand that copies the palindrome before the adjoining 4-bp repeat, and in which preferential use of the 4-bp repeats independent of the overall direction of replication implies that deletions arise during syntheses of both leading and lagging strands.     

Positive correlation between DNA polymerase alpha-primase pausing and mutagenesis within polypyrimidine/polypurine microsatellite sequences

Microsatellite DNA sequences are ubiquitous in the human genome, and mutation rates of these repetitive sequences vary with respect to DNA sequence as well as length. We have analyzed polymerase-DNA interactions as a function of microsatellite sequence, using polypyrimidine/polypurine di- and tetranucleotide alleles representative of those found in the human genome. Using an in vitro primer extension assay and the mammalian DNA polymerase alpha-primase complex, we have observed a polymerase termination profile for each microsatellite that is unique to that allele. Interestingly, a periodic termination profile with an interval size (9-11 nucleotides) unrelated to microsatellite unit length was observed for the [TC](20) and [TTCC](9) templates. In contrast, a unit-punctuated polymerase termination profile was found for the longer polypurine templates. We detected strong polymerase pauses within the [TC](20) allele at low reaction pH which were eliminated by the addition of deaza-dGTP, consistent with these specific pauses being a consequence of triplex DNA formation during DNA synthesis. Quantitatively, a strand bias was observed in the primer extension assay, in that polymerase synthesis termination is more intense when the polypurine sequence serves as the template, relative to its complementary polypyrimidine sequence. The HSV-tk forward mutation assay was utilized to determine the corresponding polymerase alpha-primase error frequencies and specificities at the microsatellite alleles. A higher microsatellite polymerase error frequency (50x10(-4) to 60x10(-4)) was measured when polypurine sequences serve as templates for DNA synthesis, relative to the polypyrimidine template (18x10(-4)). Thus, a positive correlation exists between polymerase alpha-primase pausing and mutagenesis within microsatellite DNA alleles.     

3' -> 5' Exonucleases of DNA Polymerases ε and δ Correct Base Analog Induced DNA Replication Errors on opposite DNA Strands in Saccharomyces Cerevisiae

The base analog 6-N-hydroxylaminopurine (HAP) induces bidirectional GC -> AT and AT -> GC transitions that are enhanced in DNA polymerase ε and δ 3' -> 5' exonuclease-deficient yeast mutants, pol2-4 and pol3-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases δ and ε contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC -> AT and AT -> GC transitions in a Pol(+), pol2-4 or pol3-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either pol2-4 or pol3-01, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the ura3 alleles placed into chromosome III near to the defined active replication origin ARS306 in two orientations indicated that DNA polymerases ε and δ correct HAP-induced DNA replication errors on opposite DNA strands.     

Effects of multiple yeast rad3 mutant alleles on UV sensitivity, mutability, and mitotic recombination.

A yeast strain was constructed that had a disruption of the chromosomal RAD3 gene and carried a series of centromeric plasmids with defined mutations in this gene. Using this isogenic collection, we examined sensitivity to UV radiation, spontaneous and UV radiation-induced mutagenesis, and mitotic recombination. Several alleles resulted in a marked increase in UV sensitivity. Most of these alleles were found to carry mutations located in consensus motifs for DNA helicases. Other alleles caused a modest or no increase in UV sensitivity and carried mutations in regions of the Rad3 polypeptide that are apparently not conserved. This correlation suggests that the DNA helicase activity of Rad3 protein is required for nucleotide excision repair of DNA. Some rad3 alleles conferred a marked increase in the frequency of spontaneous mutagenesis, including nonsuppressor reversion of the lys2-1 ochre mutation. These alleles also showed a good correlation with conserved DNA helicase domains, suggesting that the Rad3 DNA helicase also plays a role in the fidelity of DNA synthesis or postreplicative mismatch correction. Several rad3 mutator alleles also resulted in increased levels of mitotic recombination. Increased spontaneous mutagenesis and mitotic recombination are characteristic features of the Rem- phenotype. However, in contrast to the prototypic Rem- phenotype, the rad3 mutator alleles identified in this study did not confer inviability in the presence of mutations in the RAD50 or RAD52 gene required for strand break repair of DNA.     

Helicase-appended Topoisomerases: New Insight into the Mechanism of Directional Strand Transfer

DNA strand passage through an enzyme-mediated gate is a key step in the catalytic cycle of topoisomerases to produce topological transformations in DNA. In most of the reactions catalyzed by topoisomerases, strand passage is not directional; thus, the enzyme simply provides a transient DNA gate through which DNA transport is allowed and thereby resolves the topological entanglement. When studied in isolation, the type IA topoisomerase family appears to conform to this rule. Interestingly, type IA enzymes can carry out directional strand transport as well. We examined here the biochemical mechanism for directional strand passage of two type IA topoisomerases: reverse gyrase and a protein complex of topoisomerase IIIα and Bloom helicase. These enzymes are able to generate vectorial strand transport independent of the supercoiling energy stored in the DNA molecule. Reverse gyrase is able to anneal single strands, thereby increasing linkage number of a DNA molecule. However, topoisomerase IIIα and Bloom helicase can dissolve DNA conjoined with a double Holliday junction, thus reducing DNA linkage. We propose here that the helicase or helicase-like component plays a determinant role in the directionality of strand transport. There is thus a common biochemical ground for the directional strand passage for the type IA topoisomerases.