Multiple DNA polymerases act during homologous recombination repair in Drosophila melanogaster

• Main Author: Kane, Daniel P
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2012
• Subjects: Biology; Genetics; Molecular biology
• ISBN: 9781267409065; 1267409061

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 template 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 unclear.