Section: Research

Paul D. Kaufman, Ph.D.

Academic Role: Associate Professor

Faculty Appointment(s) In:
   Program in Gene Function and Expression
   Program in Molecular Medicine

Assembly and Function of Eukaryotic Chromosomes

Our goal is to understand how eukaryotic cells assemble chromosomes.  We are specifically interested in the coordination of DNA synthesis and chromatin assembly, because coupling these processes prevents genome rearrangements and cell cycle perturbations.  Our work is focused on the initial steps in chromosome assembly, the deposition of histones onto DNA.  We study chromosome assembly and function in yeast and human cells, using biochemical, genetic, and cell biological techniques.

Our research began with CAF-I (Chromatin Assembly Factor-I), an evolutionarily conserved heterotrimeric protein complex, which deposits histones H3/H4 onto newly replicated DNA.  We demonstrated that yeast CAF-I is important for resistance to DNA damage and builds heterochromatic and centromeric chromatin. However, CAF-I is not the only factor that that deposits H3/H4, leading to our discovery of the Hir proteins as central players in a second assembly pathway.  We are pursuing detailed biochemical characterization of CAF-I, the Hir proteins, and their shared partner, a highly conserved histone deposition protein termed Asf1.  We are also studying chromosome formation in human cells, and we have found that perturbation of CAF-I in cultured cells blocks S phase progression and activates an ATM/ATR-dependent checkpoint.

Current Projects
Biochemical and structural analysis of nucleosome assembly proteins

To facilitate precise understanding of Asf1 function, we solved the crystal structure of the highly conserved globular domain of Asf1, which mediates all functions of the full-length protein. In collaboration with the laboratory of James Berger at UC Berkeley, we determined the crystal structure of the Asf1 core domain to 1.5 Å resolution, revealing a compact immunoglobulin-like fold (see Figure).  The surface of Asf1 displays a conserved hydrophobic groove flanked on one side by an area of strong electronegative surface potential.  These regions represent potential binding sites for histones and other interacting proteins.  The structural model also allowed us to interpret recent mutagenesis studies of the human Asf1 proteins and to functionally define the region of Asf1 responsible for Hir1-dependent telomeric silencing in budding yeast.  We are currently performing a detailed structure-function analysis of Asf1 to gain insight into how this protein interacts with other histone deposition and DNA replication proteins.

Genome stability in yeast: Asf1 is required for replisome stability

Our biochemical studies of Asf1 have shown that Asf1 binds to interacts directly with Replication Factor C (RFC), the five-subunit complex that loads homotrimeric PCNA rings onto DNA during replication.  Binding of Asf1 to RFC also recruits Asf1 to DNA.  We hypothesize that this interaction is important for the role of Asf1 at the DNA replication fork.  Yeast asf1-D mutants are very sensitive to the replication inhibitor hydroxyurea (HU) and are unable to reenter the cell cycle after treatment with HU, suggesting irreversible damage.  We have found that multiple DNA replication proteins are displaced from stalled forks in asf1-D mutants, providing a molecular mechanism for the HU sensitivity.  Together, our studies demonstrate that histone deposition protein Asf1 binds RFC and is required for the stability of replication forks in vivo.  We are currently characterizing the anatomy of replication forks in asf1-D cells by a variety of techniques, including chromatin immunoprecipitation and 2D gel analysis of DNA intermediates.

Genome stability in human cells: surveillance of nucleosome assembly during S phase

To investigate the contribution of CAF-I to chromatin formation in human cells, we first designed dominant-negative inhibitors based on our mapping of the CAF-I subunit interaction domains.  Specifically, we focused on a C-terminal fragment of the human CAF-I large subunit (termed p150C) that binds the middle CAF-I subunit but not PCNA. First, we confirmed that p150C inhibits nucleosome assembly but not DNA synthesis in vitro.  In collaboration with Dr. Peter Adams, Fox Chase Cancer Center, we discovered that transient expression of p150C causes a dramatic S phase delay in human tissue culture cells.  This delay is very similar to that observed upon overexpression of the human Hir protein homolog, HIRA.  Specifically, histone H2AX becomes phosphorylated, and p53 becomes stabilized and phosphorylated on serine 15.  Importantly, the cell cycle delay caused by p150C or HIRA overexpression requires the presence of either the related ATR or ATM checkpoint kinases.  These data indicated that perturbation of nucleosome assembly results in DNA damage recognized by the ATM/ATR kinases. We hypothesize that this results from instability of DNA replication forks when rapid nucleosome assembly is absent.  To pursue studies of the role of CAF-I in genome stability, we have generated inducible siRNA cell lines that destroy CAF-I in a regulated manner.  These cells will allow us to arrest large uniform populations for further physiological and biochemical analysis.

Office: LRB 506
Phone: 508-856-5016
E-mail: Paul.Kaufman1@umassmed.edu
Keywords: Organisms - vertebrate/human cell lines, Chromosome Structure & Dynamics, Protein Acetylation and Deacetylation, Biochemistry, Organisms - yeast

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