The initiation of DNA synthesis and its regulation is a fundamental process of biology that impacts virtually every aspect of human health. Proper replication determines the fate of cells during early development and throughout adult life. DNA polymerases cannot synthesize DNA without a primer, and primase is the specialized RNA polymerase capable of de novo synthesis of short RNA primers during replication. In eukaryotes, primase functions within a heterotetrameric primase-DNA polymerase alpha (pol alpha) complex. This complex is uniquely capable of switching from the synthesis of RNA by primase to the synthesis of DNA by pol alpha. The synthesized RNA-DNA primer is required for further DNA synthesis by the major replicative DNA polymerases. In humans, the primase component consists of a small catalytic subunit (p49) and a large subunit (p58), and pol alpha is comprised of a catalytic subunit (p180) and an accessory subunit B (p70). The concerted actions of primase and pol alpha are critical for accurate genome duplication. Malfunction of primase-pol alpha complex causes global genome instability and is linked to the onset and progression of cancer and other diseases. Currently, the details for primase-pol alpha complex organization and function, including the mechanisms of unit size RNA primer synthesis and subsequent internal transfer to pol alpha are very limited. The goal of our project is to determine the structural basis of human primase-pol alpha complex function and reveal the biological consequences of alterations in this complex. To achieve our goal we will determine the mechanism of unit-length RNA primer synthesis and counting by human primase (Aim 1), the structural and functional consequences of primase integration into the pol alpha complex (Aim 2), and the mechanism of substrate switch from primase to pol alpha (Aim 3). Our studies will involve a variety of methods: X-ray crystallography, small angle X-ray scattering (SAXS), surface plasmon resonance (SPR), single molecule experiments, yeast two-hybrid and polymerase reactions assays. We also will examine the in vivo impact of mutations affecting primase-pol alpha activities on genome stability in a yeast model system.

Public Health Relevance

The knowledge of the structure and mechanism of the primase-pol alpha complex will have tremendous significance in medicine. The key advantage of our project is the focus on human replication proteins, which are poorly characterized in comparison to bacterial and archaeal counterparts. The crystal structure of the primase-pol alpha complex will provide a basis for the design of drugs targeting only viral, bacterial and possibly yeast primases and polymerases but not human primase-pol alpha.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM101167-01A1
Application #
8504521
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2013-04-01
Project End
2017-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
1
Fiscal Year
2013
Total Cost
$438,959
Indirect Cost
$138,959
Name
University of Nebraska Medical Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
168559177
City
Omaha
State
NE
Country
United States
Zip Code
68198
Gu, Jianyou; Babayeva, Nigar D; Suwa, Yoshiaki et al. (2014) Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4. Cell Cycle 13:1788-97
Shrivastava, T; Mino, K; Babayeva, N D et al. (2014) Structural basis of Ets1 activation by Runx1. Leukemia 28:2040-8
Zhang, Yinbo; Baranovskiy, Andrey G; Tahirov, Tahir H et al. (2014) The C-terminal domain of the DNA polymerase catalytic subunit regulates the primase and polymerase activities of the human DNA polymerase ?-primase complex. J Biol Chem 289:22021-34
Siebler, Hollie M; Lada, Artem G; Baranovskiy, Andrey G et al. (2014) A novel variant of DNA polymerase ?, Rev3?C, highlights differential regulation of Pol32 as a subunit of polymerase ? versus ? in Saccharomyces cerevisiae. DNA Repair (Amst) 24:138-49
Spies, Maria (2014) Two steps forward, one step back: determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers. DNA Repair (Amst) 20:58-70
Baranovskiy, Andrey G; Gu, Jianyou; Babayeva, Nigar D et al. (2014) Crystallization and preliminary X-ray diffraction analysis of human DNA primase. Acta Crystallogr F Struct Biol Commun 70:206-10
Ghoneim, Mohamed; Spies, Maria (2014) Direct correlation of DNA binding and single protein domain motion via dual illumination fluorescence microscopy. Nano Lett 14:5920-31