Cha group

Signal transduction and eukaryotic DNA replication.

We use the budding yeast S. cerevisiae to study eukaryotic DNA replication Our two main interests are:

1. the roles of signal transduction in monitoring and regulating DNA replication
2. the ways in which defects in these processes lead to chromosomal damage, genome instability, and cell death

The budding yeast gene MEC1 encodes for a signal transduction protein that is essential for viability, checkpoint regulation, and other mitotic and meiotic processes. Mec1 is also a homolog of the mammalian ATM and ATR tumor suppressors. Previously, we reported that Mec1 is required for promoting replication fork progression and that defects in this process lead to genome-wide fork stalling followed by chromosome fragmentation and cell death. Further analyses revealed that the occurrence of chromosomal breakage is confined to specific regions in the genome where the rate of replication progression is notably slow. These break-susceptible regions were named Replication Slow Zones (RSZs) and proposed to be analogous to the mammalian fragile sites. Recent studies linking the inactivation of ATR to chromosome fragmentation at a fragile site indicate that the mechanisms underlying chromosome breakage in the yeast and mammals are conserved, and that mec1 mutants are an excellent model system in which to study the genome instability and tumorigenesis occurring in ATM and ATR individuals. Our current research is focused on three major areas:

Hotspots for chromosome breakage in the eukaryotic genome

The budding yeast RSZs and the mammalian fragile sites are regions in the genome with hypersensitivity to chromosome breakage. Such break-susceptible regions are implicated in a number of important biological phenomena including genome instability, evolution, and cancer. We wish to better understand why certain regions of the genome are particularly "fragile". Specifically, we are examining the primary structure of DNA sequence, chromatin structure, and the nature of biochemical processes targeted to these regions.

Mec1 mediated replication fork progression

The formation of lethal chromosomal breaks in mec1 mutants stems from a defect in promoting replication fork progression. To better understand the precise mechanism of Mec1's action, we are utilizing genetic and biochemical approaches to identify factors that are involved in this process, and the ways in which each component is regulated by Mec1.

Roles of Mec1 in coupling chromosome morphogenesis and DNA replication

Previously, we have shown that the status of replication-dependent chromosome morphogenesis (e.g. the establishment of sister chromatid cohesion) can either lengthen or shorten the overall duration of S-phase. Based on this and other observations, we proposed the existence of an intra-S-phase regulatory network that monitors the status of chromosome morphogenesis and regulates ongoing DNA replication accordingly. We are currently exploring the possibility that Mec1 is the central regulatory component of this feedback system using mec1 mutants that exhibit either shorter or longer S-phases than the wild type parental strain.

Cellular catastrophe following Mec1 inactivation ::

Wild type (MEC1) and mutant (mec1) cells have been stained to visualize their DNA (blue). In the wild type cells, nucleus is intact and the DNA is confined within the nucleus. In mutant cells that have been committed to death, the DNA exhibits an abnormal pattern of compaction/aggregation, and is found throughout the cell.

Replication Slow Zones (RSZs) in yeast chromosome III ::

Chromosome breakage following inactivation of Mec1 mostly occurs within RSZs (filled rectangles). RSZs are ~10kb wide and occur in alternation with active replication origins (open circle) throughout the entire chromosome except for the centromeric region (filled oval). Chr III is 320 kb long; midpoint of each RSZ is as indicated.

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