CHARACTERIZING REPLICATION PROTEIN RECRUITMENT AT GAA MICROSATELLITE REPEAT IN A MAMMALIAN PRIMARY REPLICATION CYCLE

Open Access
Author:
Degiosio, Rebecca Anne
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Maria Krasilnikova, Thesis Supervisor
  • Lorraine Santy, Honors Advisor
Keywords:
  • microsatellite
  • DNA replication
  • mammalian
  • genomic instability
  • chromatin immunoprecipitation
  • quantitative PCR
  • trinucleotide repeat
  • Friedreich's Ataxia
Abstract:
The expansion of microsatellites – stretches of DNA consisting of repeating 2-5 nucleotide motifs – has been implicated as the key precipitant to a host of genetic disorders as well as general genomic instability. The mechanism of such expansion, however, remains unclear. It has been previously demonstrated that mammalian cells undergo a distinct primary cycle of DNA replication which is particularly susceptible to replication stalling by non- canonical DNA structures formed within repetitive genomic regions. It is unknown if this primary replication cycle plays any role in facilitating microsatellite repeat expansion. Herein, we investigate patterns of replication protein recruitment during primary and subsequent replication cycles in regions surrounding a trinucleotide repeat. SV40-origin based plasmids containing GAA57 repeat in two orientations or no repeat transfected into mammalian cells were used as models of DNA replication. Chromatin immunoprecipitation (ChIP) was performed during both primary and subsequent cycles using antibodies to polymerase alpha (pol α)/primase, MCM4, and polymerase zeta (pol ζ) proteins. Results were analyzed by regular PCR and quantitative PCR (qPCR). We demonstrate that pol α and MCM4 are more strongly recruited in repeat-containing plasmids than in control plasmid during the primary replication cycle specifically. We propose that these proteins’ recruitment in this early stage results from elevated non-canonical DNA structure formation in the presence of repeat, permitted by underdeveloped chromatin structure. The polymerase may then initiate the unstable primary cycle of replication, leading to possible repeat expansion and increased genomic instability.