Loss or alteration of p53 function occurs in about half of all human cancers. The ongoing focus of our research continues to be understanding how p53 and the mismatch repair (MMR) proteins interact with DNA possessing simple lesions or large damage-containing structures and signal damage. Recently our studies revealed a surprising overlap in the binding of p53 and the MMR proteins to complex DNA structures as well as some simple lesions, in particular bulged bases and some base/base mismatches. Further, there may be synergistic interactions between p53 and MSH2-MSH6. By using a combination of biochemical tools and direct electron microscopic (EM) visualization, we are able to approach questions which cannot be addressed easily by other experimental means. One poorly understood question is how cells sense the presence of large damaged DNA structures, or potentially lethal DNA entanglements. Examples include large intertwined DNAs– which if not resolved might lead to chromosome loss, Holliday junctions containing clusters of bulged bases on one arm, or replication forks with damage on a newly synthesized strand. These structures must be resolved or repaired prior to chromosome segregation, and thus cell cycle checkpoints must be in place to monitor their presence. It is likely that p53 participates in some of these checkpoints. Our long term goal in this project is to construct large DNA molecules with multiple structural features, for example replication forks with specific lesions near by, and then to ask how p53 and the MMR proteins together with other cellular proteins interact with these structures. This is an ongoing project and we will continue our studies on the binding of p53 to simple lesion-containing DNAs, and initiate studies on the remodeling of complex DNAs containing multiple structural features by p53, the MMR proteins and other cellular factors.

Stemming from the discovery of p53 consensus binding sites in DNA near p53 regulated genes, and the finding that p53 is a potent transactivator, it was assumed that all p53 dependent functions would be mediated via transcriptional mechanisms. Over the past several years, however, a growing body of evidence has accumulated suggesting a more direct role of p53 in some processes. It is known that p53 binds single-stranded (ss) DNA and double-stranded (ds) DNA ends, both typical products of DNA damage. We then showed that p53 forms stable complexes at sites of insertion/deletion mismatches (bulged or looped out bases) in DNA, a finding which was surprising at the time and suggested that p53 may participate directly in signaling the presence of DNA damage. Recently we used filter binding and gel shift assays to measure the binding of p53 to all possible single base/base mismatches in otherwise simple linear DNA (see Degtyareva et al below).

There are a large number of DNA repair factors that are now known to interact alone and in concert with other repair and telomere-related proteins on damaged DNA. We have isolated many of these factors and have others available via collaborations. These include Rad51, the Rad51 paralogs, the WRN and BLM helicases, polymerase beta, and BRCA2. Understanding how these proteins alone and in combination interact at sites of DNA damage is critical to our understanding of genomic repair and genomic toxicology. In this regard, EM is unique in being able to answer biochemical and structural questions that would be nearly impossible to answer by any other approach. Our laboratory is also able to engineer and build large complex DNA templates, kilobases in size which have replication forks, Holliday junctions, and other structures. These are unique templates for the EM studies and are much more relevant to events in the cell than tiny DNAs generated from oligonucleotides.

Selected References:

  • Suman Lee, Brian Elenbaas, Arnold Levine and Jack Griffith. p53 and its 14 kDa C terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. Cell 81: 1013-1020, 1995.
  • Gerald T. Marsischky, Suman Lee, Jack Griffith, and Richard D. Kolodner. Saccharomyces cerevisiae MSH2/6 complex interacts with Holliday junctions and facilitates their cleavage by phage resolution enzymes. J. Biol. Chem. 274. 7200-7206. 1999.
  • Natalya Degtyareva, Deepa Subramanian, and Jack D. Griffith. Analysis of the binding of p53 to DNAs containing mismatched and bulged bases. J. Biol. Chem. 276: 8778-8784. 2001.
  • Rachel Stansel, Deepa Subramanian, and Jack D. Griffith. p53 Binds Telomeric Single Strand Overhangs and t-loop Junctions in Vitro. J. Biol. Chem. 277, 11,625-11, 628, 2002.
  • Keziban Unsal-Kacmaz, Alexander Makhov, Jack D. Griffith, and Aziz Sancar. Preferential binding of ATR protein to UV-damaged DNA. Proc. Natl. Acad. Sci. USA. 99: 6673-6678, 2002.
  • Deepa Subramanian and Jack D. Griffith. Modulation of p53 binding to Holliday junctions and 3-cytosine bulges by phosphorylation events. Biochemistry. 44: 2536-2544, 2005.
  • Thorslund T, McIlwraith MJ, Compton SA, Lekomtsev S, Petronczki M, Griffith JD, West SC.  The breast cancer tumor suppressor BRCA2 promotes the specific targeting of RAD51 to single-stranded DNA. Nat Struct Mol Biol. 2010;  10:1263-5. PMID: 20729858
  • Rass U, Compton SA, Matos J, Singleton MR, Ip SC, Blanco MG, Griffith JD, West SC. Mechanism of Holliday junction resolution by the human GEN1 protein. Genes Dev. 2010  24(14):1559-69. PMID: 20634321
  • Compton SA, Ozgür S, Griffith JD. Ring-shaped Rad51 paralog protein complexes bind Holliday junctions and replication forks as visualized by electron microscopy.  J Biol Chem. 2010 285(18):13349-56..PMID: 20207730