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.
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.
|Baranovskiy, Andrey G; Duong, Vincent N; Babayeva, Nigar D et al. (2018) Activity and fidelity of human DNA polymerase ? depend on primer structure. J Biol Chem 293:6824-6843|
|Baranovskiy, Andrey G; Siebler, Hollie M; Pavlov, Youri I et al. (2018) Iron-Sulfur Clusters in DNA Polymerases and Primases of Eukaryotes. Methods Enzymol 599:1-20|
|Baranovskiy, Andrey G; Babayeva, Nigar D; Zhang, Yinbo et al. (2017) Comment on ""The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport"". Science 357:|
|Baranovskiy, Andrey G; Gu, Jianyou; Babayeva, Nigar D et al. (2017) Crystal structure of the human Pol? B-subunit in complex with the C-terminal domain of the catalytic subunit. J Biol Chem 292:15717-15730|
|Baranovskiy, Andrey G; Tahirov, Tahir H (2017) Elaborated Action of the Human Primosome. Genes (Basel) 8:|
|Mertz, T M; Baranovskiy, A G; Wang, J et al. (2017) Nucleotide selectivity defect and mutator phenotype conferred by a colon cancer-associated DNA polymerase ? mutation in human cells. Oncogene 36:4427-4433|
|Zhang, Yinbo; Baranovskiy, Andrey G; Tahirov, Emin T et al. (2016) Divalent ions attenuate DNA synthesis by human DNA polymerase ? by changing the structure of the template/primer or by perturbing the polymerase reaction. DNA Repair (Amst) 43:24-33|
|Baranovskiy, Andrey G; Zhang, Yinbo; Suwa, Yoshiaki et al. (2016) Insight into the Human DNA Primase Interaction with Template-Primer. J Biol Chem 291:4793-802|
|Boehm, E M; Subramanyam, S; Ghoneim, M et al. (2016) Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy. Methods Enzymol 581:105-145|
|Boehm, Elizabeth M; Powers, Kyle T; Kondratick, Christine M et al. (2016) The Proliferating Cell Nuclear Antigen (PCNA)-interacting Protein (PIP) Motif of DNA Polymerase ? Mediates Its Interaction with the C-terminal Domain of Rev1. J Biol Chem 291:8735-44|
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