Sue Jinks-Robertson, PhD
Director, Cell and Molecular Biology Program
384 CARL Building
Box 3020 DUMC
Durham, N.C. 27710
Phone: (919) 681-7273
Fax: (919) 684-6033
DNA repair and surveillance mechanisms are essential for the maintenance of eukaryotic genome integrity. In humans, these mechanisms protect somatic tissue from the accumulation of the genetic changes associated with cancer and ensure that informational content is stably transmitted from one generation to the next. My laboratory uses the yeast Saccharomyces cerevisiae as a model genetic system to understand the pathways/mechanisms that regulate mitotic genome stability. There are two major areas of focus within the lab: (1) defining molecular mechanisms of homologous recombination and (2) understanding mechanisms that contribute to transcription-associated genetic instability.
Mechanisms of homologous recombination
Mitotic recombination is an essential repair process that fills single-strand gaps and repairs double-strand breaks (DSBs). Though most recombination likely occurs between identical sister chromatids and hence is of no genetic consequence, recombination also can involve homologous chromosomes, leading to loss of heterozygosity and expression of recessive markers. In addition, interactions between dispersed repeated sequences can generate a variety of detrimental genome rearrangements including deletions/duplications, inversions and translocations. We have developed novel molecular approaches and tools that allow us to precisely follow the swapping of single strands between duplex DNAs during the repair of defined DSBs. This is accomplished through the use of substrates with engineered single-nucleotide polymorphisms (SNPs) at ~50 bp intervals. Strand exchange generates mismatch-containing “heteroduplex” DNA (hetDNA), the length and position of which can be determined by sequencing recombination products. The position of hetDNA in individual recombinants can be used to infer the underlying molecular mechanism of recombination. Alterations in hetDNA patterns in defined mutant backgrounds provide molecular detail that cannot be obtained using more traditional methods. Our molecular analyses have confirmed, for example, that most mitotic recombination occurs via the synthesis-dependent strand-annealing pathway and have clarified how individual helicases regulate recombination outcome.
The basic framework for mapping hetDNA was developed using a transformation-based assay in which a broken plasmid is repaired using a SNP-containing chromosomal template. More recent work has employed chromosomal substrates, one of which contains a inducible DSB, and has confirmed basic hetDNA patterns observed in the transformation-based assay. To complement analysis of DSB-initiated recombination, substrates are being developed that will allow the molecular features of recombination initiated either spontaneously or by a defined single-strand nick to be ascertained. Finally, computational tools are being developed to sequence recombination events en masse using the high throughput, single-molecule PacBio platform.
Transcription and genome stability
Because the DNA metabolic processes of transcription, replication, recombination and repair are not temporally separated, one process has the potential to influence the occurrence of another. Our work has demonstrated that the stability of DNA is related not only to its primary sequence, but also is influenced by its level of transcription. Reporters fused to the highly inducible pGAL or doxycycline-regulated pTet promoter have been used to study how transcription locally stimulates mutagenesis, a phenomenon referred to as transcription-associated mutagenesis or TAM. We have discovered that there are multiple sources of TAM, including elevated damage accumulation and increased substitution of uracil for thymine in the underlying DNA template. The major source of TAM in an unbiased forward mutation assay, however, is due to activity of Topoisomerase 1 (Top1), the enzyme that removes transcription-associated supercoils. Top1-dependent mutations have a distinctive molecular signature comprised of 2-5 bp deletions that eliminate a single repeat unit of a sequence repeated 2-4 times. Genetic studies suggest that deletions reflect either (1) the mutagenic processing of a trapped Top1 cleavage complex or (2) the sequential action of Top1 when the initial incision occurs at the site of a ribonucleotide monophosphate embedded in DNA. Current work is further exploring the mechanisms of Top1-dependent mutagenesis.
In addition to TAR studies, we are collaborating with Tom Petes to determine how and where persistent base pairing of the RNA transcript with the DNA template (an R-loop) affects recombination on a genome-wide scale. R-loops specifically accumulate in an RNaseH-defective strain, which is unable to degrade the RNA component of R-loops. Using a selective system that genetically detects loss-of heterozygosity (LOH) on a single yeast chromosome, we have found that R-loop persistence elevates recombination ~10 fold. Microarrays that monitor SNP status across the entire yeast genome are being used to detect R-loop associated LOH on a genome-wide scale.