The goal of this research project is to determine the three-dimensional structure of the human DNA polymerase alpha primase complex (pol-prim) by cryo-electron microscopy methods. Pol-prim is essential for initiating synthesis of DNA, because it is uniquely able to synthesis nascent DNA when presented with a single stranded DNA template. Pol-prim synthesizes the short RNA/DNA oligonucleotide that is required to prime elongation synthesis, which is performed by the polymerase delta in conjunction with the processivity factor PCNA (Proliferating Cell Nuclear Antigen). In addition to initiating DNA replication, pol-prim activity is require to establish the S-M checkpoint, which ensures replication of chromosomes occurs before they are segregated to daughter cells. It is also an important regulator of telomere length, DNA damage responses, and chromatin formation. Mutation of proteins in these pathways may be a critical step in the progression of tumorigenesis, allowing for the multiple mutations associated with cancer cells. The role of pol-prim in such a wide array of genome maintenance activities makes it an exceptionally interesting therapeutic target. As a first step in realizing this goal and to understand its diverse roles in chromatin metabolism, we propose a detailed structural analysis of pol-prim and its relevant complexes in SV40 genome replication initiation by cryo-electron microscopy.
