Control of Programmed Replication fork Arrest by Chromosome Kissing

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DESCRIPTION (provided by applicant): Major DNA transactions such as transcription, development, X-chromosome inactivation and chromosomal translocations in cancer cells were reported to be controlled by chromosome to chromosome contacts called "chromosome kissing" mediated by a myb-like terminator protein called Reb1. We have recently shown in a paper to be published in Cell that chromosome kissing in fission yeast also controls replication fork movement by controlling sequence-specific, physiologically programmed fork arrest. This finding raises some major questions: (i) which proteins are involved in controlling chromosome kissing and what are their mechanisms of action?; (ii) what is the mechanism of programmed fork arrest? In the context of addressing the first question, we have discovered that two chromatin remodeling proteins of fission yeast modulate programmed fork arrest. The implication is that these remodelers do this by modulating chromosome kissing and nucleosome disposition about the replication termini. One of the goals of this proposal is to discover in a genome-wide search the proteins that modulate chromosome kissing and try to understand their mechanism of action. Another important goal of this proposal is to uncover the mechanism of action of programmed fork arrest by testing the hypothesis that this happens by unidirectional arrest of the MCM2-7 helicase. Programmed fork arrest controls other chromosome transactions such as recombination, gene silencing and transcriptional passage and is at the interphase of replication and other processes. Finally, experiments are proposed to localize genome-wide Reb1-dependent replication termini. The objectives of this goal is to test two hypotheses: (i) naturally occurring, noncanonical weak sites are rendered functional by chromosome kissing and/or DNA looping -dependent interaction with strong canonical sites and that is one function of these long range protein- DNA interactions; (ii) a function of looping-dependent (or independent) fork arrest is to prevent interference between replication and transcription. PUBLIC HEALTH RELEVANCE: The existing models of replication control are two dimensional consisting of proteins that interact with sequences of a single chromosome at a time to initiate replication, promote fork progression etc. Our recent work shows that replication control is 3 dimensional. This application proposes to investigate further this mechanism that is potentially relevant to human diseases such as cancer.