Regulation of replication-coupled DNA maintenance

Multiple genomic maintenance pathways have evolved in all living organisms that safeguard the integrity of the genome. Each pathway is typically specialized in handling specific types of DNA damage, such as single- and double-stranded breaks, base damage and mismatches. A novel maintenance pathway, defined by the Fanconi anemia (FA) proteins, has been discovered to operate exclusively in vertebrates. This ?FA pathway? appears to be indispensable for the correct replication of DNA, presumably by resolving blocked replication forks. Our lab has played a leading role in identifying the thirteen proteins that participate in this pathway as well as their molecular associations. Although the FA proteins and protein complexes have the intrinsic capacity to maintain genomic stability, it is key that they are directed by regulatory pathways. What is critically missing at the current stage of understanding is how the various FA proteins are regulated to function properly and this will be the focus of our project. We hypothesize that posttranslational modification may represent an important mechanism in the regulation of the FA pathway and want to assess whether and how the pathway is regulated by these modifications. This proposal focuses mainly on FANCM, -J, and -N, which appear to occur in modified forms. We will characterize the modifications by mass spectrometry analysis of purified proteins obtained from cellular extracts. Follow-up studies will focus on the conditions that elicit the modifying response, the enzymes that cause the modifications and the consequences of the modifications for the activities of the proteins involved.