Helicase activation and progression through chromatin
During my PhD, I have studied helicase activation in vivo in the budding yeast using a polymerase alpha conditional mutant. By blocking priming, the first step in DNA replication, I have captured the active helicase and DNA unwinding prior to the onset of strand synthesis. Using chromatin profiling along with ChIP-seq for specific replication factors, I have determined that the active CMG complex unwinds DNA up to 1 kilobase from the origin before stalling, disrupting approximately six nucleosomes. Intriguingly, the helicase stalls as it moves out of the origin region, which is characterized by an AT-rich sequence in yeast. This led us to propose that the CMG likely stalls because it is less processive in the absence of replication, and therefore has difficulty unwinding sequences that have a higher thermodynamic stability. Our current model for helicase activation in the absence of replication in budding yeast origins is shown on the right.
MNase (micrococcal nuclease) chromatin profiling
In eukaryotic organisms, the genome exists within a structure called chromatin. The most basic unit of chromatin mainly consists of nucleosomes, which are ~150 base pairs of DNA wrapped around an octamer of histone proteins. Other proteins also bind to the DNA and regulate its usage. DNA repair, replication, and transcription all interact with chromatin structure to access the DNA double helix. To study these dynamic processes, our research group has developed a factor-agnostic chromatin profiling assay that allows us to determine the positions of nucleosomes and DNA-bound proteins, such as transcription factors. In this assay, we digest chromatin with MNase, and recovered fragments of DNA protected from MNase digestion. These fragments were protected by some factor associated with the DNA, the identify of which we can infer based on fragment length. An example of the data produced by this assay is shown on the left. I have worked on methods to visualize MNase datasets and quantify chromatin dynamics across genomic loci.
Publications
Rachel Hoffman and David MacAlpine. Disruption of origin chromatin structure by helicase activation in the absence of DNA replication. 2021. bioRxiv. https://doi.org/10.1101/2021.03.17.435814
Anna Boudores, Jacob Pfeil, Elizabeth Steenkiste, Rachel Hoffman, Sara Wilkes, Sarah Higdon, and Jeffrey Thompson. 2017. A novel histone crosstalk pathway important for regulation of UV-induced DNA damage repair in Saccharomyces cerevisiae. Genetics 206:1389-1402. https://doi.org/10.1534/genetics.116.195735