The Alani lab use interdisciplinary and collaborative approaches to study roles for DNA mismatch repair factors in maintaining genome stability during vegetative growth and in meiosis. In humans, mutations in DNA mismatch repair genes correlate with a predisposition to a hereditary form of colorectal cancer (~2 to 7% of all colon cancers) and infertility, underscoring the importance of obtaining new mechanistic understandings of mismatch repair and diagnosis tools. We use baker’s yeast for our work, but also collaborate with mouse geneticists on the Cornell campus. We have three major projects listed below.
1. Identifying mechanistic roles for EXO1 and MLH1-MLH3 in forming interference-dependent crossovers in meiosis.
Crossing over between chromosome homologs is critical for their segregation in meiosis to form gametes. We developed a model to explain how recombination intermediates (specifically double Holliday Junctions) are resolved in meiosis in a biased manner to form crossovers. This model is based on our studies showing that the EXO1 protein protects DNA nicks from ligation to promote crossover formation.
We hypothesize that this protection promotes subsequent resolution by the MLH1-MLH3 endonuclease to form crossovers. We are currently testing this model using genetic and bioinformatic methods with the goal of obtaining a better mechanistic understanding for how crossover resolution occurs.
For more information, please see: Gioia et al (2023), Singh et al (2021), Toledo et al (2019), Manhart et al (2017)
2. Analyzing yeast strains containing incompatible combinations of MLH1 and PMS1 MMR alleles.
We identified a genetic incompatibility involving the MLH1 and PMS1 DNA mismatch repair genes in progeny obtained by crossing different strains of baker’s yeast that are 0.7% nucleotide sequence divergent.
One of the four MLH1-PMS1 combinations confers a mutator phenotype. Our work supports a model in which variance in mutation rate contributes to adaptation to stress conditions through the acquisition of beneficial mutations, with high mutation rates leading to long-term fitness costs buffered by mating or eliminated through natural selection. This work is especially relevant to understanding how fungi become pathogenic.
Our current work is focused on understanding how incompatibilities arise in the DNA mismatch repair genes and how they can be eliminated during adaptation to stress.
For more information, please see: Furman et al (2021), Raghavan et al (2019), Raghavan et al (2018), Demogines et al (2008)
3. Using genetic and biochemical approaches we are examining how chromatin environment affects the stability of recombination intermediates.
We are interested in understanding how factors involved in chromosome architecture work with DSB repair proteins to maintain genome integrity.
Our previous research in this area has highlighted the effect that histone chaperones Asf1, Caf-1, and Rtt106, as well as the histone deacetylase Sir2 act to promote efficient repair, even when utilizing non-homologous donor templates during HR. Our current area of investigation involves the silent chromatin regulator/ deacetylase complex SIR2/3/4, and how it might act during slow-to-repair HR events. Our hypothesis is that the SIR complex acts to physically stabilize D-loop structures at the nuclear pore, promoting efficient repair of DSBs repaired through slow, laborious processes such as Break Induced Replication.
Another area of investigation involves exploring the architectural changes which occur during HR repair events, and whether these architectural changes differ when cells utilize homologous or divergent repair templates.