Multiple DNA polymerases act during homologous recombination repair in Drosophila melanogaster.
Abstract: Proper repair of DNA double-strand breaks is necessary for
maintenance of genomic stability and prevention of genetic disorders. Breaks are repaired
by two canonical pathways: non-homologous end joining and homologous recombination (HR). HR
is an important double-strand break repair mechanism as it is considered an error-free
pathway. During HR, the broken strand invades a homologous... read moretemplate and synthesizes nascent
complementary DNA. This ensures retention of information that may have been lost due to the
damage event. Proteins involved for template invasion are well understood. However, the DNA
polymerases responsible for nascent synthesis during HR and how they are recruited and
coordinated at sites of double-strand breaks is not clear. Evidence exists for the use of
both replicative DNA polymerases as well as specialized translesion synthesis (TLS)
polymerases. We used a gap repair assay in Drosophila to determine how different polymerase
types are utilized during HR repair synthesis. Our results indicate that both replicative
and TLS polymerases compete for access to HR intermediates. Specifically, the TLS
polymerase Rev1 is primarily recruited to regions of template invasion and serves to
recruit other TLS polymerases (polymerase zeta) for HR initiation synthesis. Rev1 also acts
to block access of HR intermediates to processive replicative polymerase delta and end
joining proteins. In the absence of Rev1, repair synthesis is quite extensive and requires
the pol32 subunit of polymerase delta. We next wanted to determine if this processive DNA
synthesis was aided by other repair proteins. In replication, DNA polymerase clamps are
used to tether a polymerase to its template and increase its processivity. We again
utilized a gap repair assay to assess functions of two clamps during HR: PCNA (used during
replication) and 9-1-1 (used in response to DNA damage). Preliminary data suggests that
PCNA is utilized for HR synthesis, aiding processivity of various polymerase types.
Additionally, 9-1-1 is also important for HR repair, appearing to act as a signaler to
activate checkpoints and direct repair of double-strand breaks towards an HR pathway. This
study helps illuminate the mechanism of HR synthesis in metazoans that was previously
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Biology.
Advisor: Mitch McVey.
Committee: Catherine Freudenreich, Juliet Fuhrman, Kelly McLaughlin, and Lorraine Symington.
Keywords: Biology, Molecular biology, and Genetics.read less