Susan F. Cotmore

My research interests are primarily focused on the mechanisms used by the murine parvovirus MVM to replicate its unusual linear, single-stranded, DNA genome. Initiation of DNA replication at both the viral replication origins, and subsequent resolution of the viral telomeres, has been reconstructed in human and murine cell extracts supplemented with the viral initiator protein NS1. Cloned, palindromic junction fragments from concatemeric, duplex replicative intermediate forms of MVM are used as substrates for resolution, and these have been mutated and recombined to explore the structures and sequences involved in the recognition of the origin. NS1 initiates replication by introducing a site and strand-specific, single-strand nick, and itself becomes covalently attached to the 5' end of the DNA at this site. Possessing ATPase and helicase activity, it then remains in the replication fork where it mediates subsequent steps in the resolution process.

Characterizing the unique chromatin assembled during parvoviral DNA replication.

The parvoviral DNA genome is unique in the biosphere, in that it is both single-stranded and linear. Despite this alien structure, the virus inveigles its host to replicate the viral chromosome to high copy numbers, instead of the cellular genome, during the S-phase following infection. In doing so, it generates intermediates that comprise long single strands, displaced by exclusively leading strand synthesis at the viral fork. These structures strongly trigger host DNA damage responses that we presume the virus must evade. Part of the virus' replication strategy involves the elaboration of a unique form of chromatin, which ChIP analysis suggests incorporates both cellular histones and many copies of NS1, the major viral non-structural protein. Indeed, NS1 binds rather promiscuously to degenerate sequence blocks represented some 40 times through the viral genome. Insertion of transgene sequences, which do not contain NS1 binding sites, is quite deleterious to the replication and packaging of parvoviral vectors. This rotation project explores the construction of an otherwise wildtype viral genome rendered artifically devoid of NS1 binding sites throughout its entire capsid gene. 2D gel electrophoresis and nuclease protection assays will be used to look for stalling or pausing of replication forks through the capsid region, and to characterize packaging intermediates generated by mutant, compared to wildtype, virus.