Our Science – Lichten Website

Michael Lichten, Ph.D.

Laboratory of Biochemistry and Molecular Biology Head, DNA Recombination in Yeast Senior Investigator Laboratory of Biochemistry 37 Convent Drive Building 37, Room 6124, MSC 4260 Bethesda, MD 20892-4260 Phone:   301-496-9760 Fax:   301-402-3095 E-Mail:   lichten@helix.nih.gov

Biography

Dr. Michael Lichten is a research microbiologist in the Microbial Genetics and Biochemistry Section of this laboratory. He received his Ph.D. in 1982 from the Massachusetts Institute of Technology and received his research training with Dr. Maurice S. Fox and Dr. James E. Haber. He joined this laboratory as a senior staff fellow in 1987, became a senior investigator in 1995, and became a member of the Senior Biomedical Research Service in 2000.

Research

Mechanism and Control of Meiotic Recombination

We study genetic recombination and chromosome structural changes that occur during meiosis and DNA damage repair, using budding yeast as a model system. Our goal is a description of the molecular steps of meiotic recombination, and how these molecular events integrate with the changes in chromosome structure and with the cell cycle transitions that occur during meiosis and during the mitotic cell cycle.

Initiation of meiotic recombination

Meiotic recombination is initiated by double-strand DNA breaks (DSBs), formed by the type 2 topoisomerase paralog Spo11p in a mechanism that is universal among eucarotes. We are interested in factors that control where and when DSBs occur. Chromatin structure is the primary determinate of where DSBs form, but other factors also determine where, when and how frequently breaks form. Features of chromosome struture, such as telomeres and centromeres, control the overall level of DSBs in their vicinity, and the time at which a chromosome region replicates determines when DSBs occur in that region. Previous studies in budding yeast had used mutants (rad50S and sae2) that trap a covalently-linked Spo11-DNA complex, and indicated that DSBs are found mainly in ca. 70-100 kb 'hot' regions that are separated by 'cold' regions of similar size.

We have recently applied microarray-based analysis to map DSBs by an alternative method, which detects single-strand DNA associated with processed breaks in wild-type and in dmc1 mutants, which form and process DSBs but which do not repair them. The picture emerging from these studies stands in stark contrast with the prevailing view, derived from rad50S and sae2 mutants. Instead of alternating cold/hot regions of 50-100kb, we find that DSB sites show a much more even distribution across the genome, a finding consistent with Southern blot-based measures of DSB frequencies and with genetic measures of meiotic recombination (see picture 2 in Gallery).

As part of this project, we are developing analytical and computational tools to compare genome-wide distributions of events (such as DSBs) and to allow quantitative correlative comparison of distributions at different scales. These tools will be applied to genome-wide studies of chromosome composition and of differnt molecular events of chromosome metabolism, as well as to studies of the dynamics of these features over evolutionary time.

Regulation of meiotic recombination intermediate formation and resolution

The Holliday junction is thought to be a central intermediate in the recombination events that occur during DSB repair. We previously showed that the Holliday junction-containing molecules that form during meiosis are predominantly precursors to crossover recombinants; most noncrossover recombinants arise by a different, earlier pathway. We are interested in identifying the activities resonsible for the formation and resolution of these intermediates, and the regulatory circuits that integrate these processes with the meiotic cell cycle.

We had previously shown that noncrossover and crossover recombination are regulated differently during meiosis. Mutants lacking NDT80 (a meiosis-specific transcription factor responsible for transcription of ~200 genes) and in CDC5 (the yeast homolog of the mammalian polo-like kinase PLK1) arrest in meiosis with unresolved Holliday junction intermediates and very few crossovers, while noncrossover heteroduplex products are formed at normal levels and with normal timing. Ndt80 is required for meiotic transcription of CDC5, indicating that the crossover-deficiency in ndt80 mutants may be due, at least in part, on a failure to express polo-like kinase, which also modulates cohesin binding to chromosome arms and promotes the mono-orientation of kinetochores at the first meiotic division. Using an inducible CDC5 gene construct in an ndt80 mutant background, we showed that CDC5 is the only member of the Ndt80 regulon required for Holliday junction resolution. Current efforts are aimed at identifying the targets of this kinases that are directly involved in driving recombination intermediate resolution as crossovers.

We also examined the role of Sgs1, the budding yeast homolog of the mammalian BLM helicase, which has been suggested to have anti-recombination activity. Although mutants lacking the Sgs1 helicase domain show no striking alteration in meiotic crossover or noncrossover recombination, loss of the Sgs1 helicase does restore crossover recombination and synapsis of homologous chromosomes to mutants lacking components of the synaptonemal complex, a meiosis-specific structure that tightly pairs homologous chromosomes and that is necessary for normal levels of crossover recombination (see picture 1 in Gallery). This identifies the Sgs1 helicase as a potent anti-recombinator whose action is blocked by meiosis-specific chromosome proteins and structures.

We have recently completed studies examining the interplay between Sgs1 and the Mus81/Mms4 endonuclease, which has been suggested as a Holliday junction resolvase. Budding yeast lacking the Sgs1 helicase and the Mus81/Mms4 endonuclease are inviable, and indirect studies implicate homologous recombination gone awry as the cause of death. We showed that mutants lacking both enzymes have profound defects in meiotic recombination intermediate metabolism and crossover (CO) formation. Recombination intermediates (joint molecules; JMs) accumulate in these cells, many with structures that are infrequent in wild type cells. These JMs persist, preventing nuclear division. Using an inducible expression system, we restored Mus81 or Sgs1 to sgs1 mus81 cells at a time when JMs are forming. Mus81 expression did not prevent JM formation, but restored JM resolution, CO formation, and nuclear division. In contrast, Sgs1 expression reduced the extent of JM accumulation. These results indicate that Sgs1 and Mus81/Mms4 collaborate to direct meiotic recombination towards interhomolog interactions that promote proper chromosome segregation, and also indicate that Mus81/Mms4 promotes JM resolution in vivo.

The work described here was done in collaboration with: Valerie Borde and Alain Nicolas, Insitut Curie, France; Beth Rockmill and Shirleen Roeder, Yale University; Somantika Datta, University of Maryland.

This page was last updated on 3/13/2009.