Professor of Biochemistry & Biophysics, UNC-CH
PHD - University of Toronto
HONORS & AWARDS
V(D)J Recombination And Double Strand Break Repair
Non-homologous end-joining (NHEJ, end joining) pathway has an important role in all cell types repairing chromosomes with double strand breaks. NHEJ is also essential for efficient resolution of chromosome break intermediates during V(D)J recombination, a lymphocyte specific process required for assembly of the immune system’s antigen specific receptors. We use diverse molecular biological approaches in an effort to understand both how end joining works and why it sometimes does not.
NHEJ is unique among DNA repair pathways in that it attempts repair without an intact template to instruct accurate replacement of damaged DNA. We’ve shown you use at least three different DNA polymerases, each with varying dependency on an intact template, to get around this problem. We are continuing to work with Dr. Kunkel’s group at NIEHS to understand the structural basis for different template dependencies.
Given the problems NHEJ faces with accuracy, we are also working to understand why, outside of V(D)J recombination, you’d risk doing NHEJ at all. We are testing the following ideas: (unlike alternate repair pathways)…
1) NHEJ is fast and cheap: it doesn’t need to do much DNA synthesis. This might explain why NHEJ is most important in non-dividing/slowly dividing cells, where synthesis can’t be easily done.
2) NHEJ does not involve disruption of a lot of adjacent chromatin, allowing you to keep the epigenetic information stored in the chromatin near breaks intact. In contrast, alternate pathways involve extensive nucleosome remodeling, and even nucleosome eviction.
We’re also interested in how V(D)J recombination makes the mistakes that lead to lymphoid malignancy. V(D)J recombination involves the popping out of a large segment of your receptor locus, and the mechanism by which you pop this segment out is similar to the way transposons move around their host genomes. We’ve recently shown that the similarity to transposition does not end with the “popping out” step: like transposition, the enzymes responsible for excising DNA segments during V(D)J recombination also re-integrate the excised DNA into some other location in your genome. Is the target for re-integration random? Might re-integration explain the origin of some lymphoid malignancies?
Map of mouse genome with aberrant recombination sites marked by triangles; red triangles denote transpositions.
- Burkhalter MD, Roberts SA, Havener JM, Ramsden DA. Activity of ribonucleotide reductase helps determine how cells repair DNA double strand breaks. DNA Repair (Amst). 2009 Nov 2;8(11):1258-63. Epub 2009 Aug 26. PubMed PMID: 19713159; PubMed Central PMCID: PMC2763971.
- Garcia-Diaz M, Bebenek K, Larrea AA, Havener JM, Perera L, Krahn JM, Pedersen LC, Ramsden DA, Kunkel TA. Template strand scrunching during DNA gap repair synthesis by human polymerase lambda. Nat Struct Mol Biol. 2009 Sep;16(9):967-72. Epub 2009 Aug 23. PubMed PMID: 19701199; PubMed Central PMCID: PMC2767187.
- Zhou T, Akopiants K, Mohapatra S, Lin PS, Valerie K, Ramsden DA, Lees-Miller SP, Povirk LF. Tyrosyl-DNA phosphodiesterase and the repair of 3'-phosphoglycolate-terminated DNA double-strand breaks. DNA Repair (Amst). 2009 Aug 6;8(8):901-11. Epub 2009 Jun 7. PubMed PMID: 19505854; PubMed Central PMCID: PMC2763370.
- Bukowy Z, Harrigan JA, Ramsden DA, Tudek B, Bohr VA, Stevnsner T. WRN Exonuclease activity is blocked by specific oxidatively induced base lesions positioned in either DNA strand. Nucleic Acids Res. 2008 Sep;36(15):4975-87. Epub 2008 Jul 25. PubMed PMID: 18658245; PubMed Central PMCID: PMC2528166.
- Mueller GA, Moon AF, Derose EF, Havener JM, Ramsden DA, Pedersen LC, London RE. A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda. DNA Repair (Amst). 2008 Aug 2;7(8):1340-51. Epub 2008 Jun 26. PubMed PMID: 18585102; PubMed Central PMCID: PMC2583787.
- Davis BJ, Havener JM, Ramsden DA. End-bridging is required for pol mu to efficiently promote repair of noncomplementary ends by nonhomologous end joining. Nucleic Acids Res. 2008 May;36(9):3085-94. Epub 2008 Apr 8. PubMed PMID: 18397950; PubMed Central PMCID: PMC2396419.
- DeRose EF, Clarkson MW, Gilmore SA, Galban CJ, Tripathy A, Havener JM, Mueller GA, Ramsden DA, London RE, Lee AL. Solution structure of polymerase mu's BRCT Domain reveals an element essential for its role in nonhomologous end joining. Biochemistry. 2007 Oct 30;46(43):12100-10. Epub 2007 Oct 4. PubMed PMID: 17915942; PubMed Central PMCID: PMC2653216.
- Roberts SA, Ramsden DA. Loading of the nonhomologous end joining factor, Ku, on protein-occluded DNA ends. J Biol Chem. 2007 Apr 6;282(14):10605-13. Epub 2007 Feb 8. PubMed PMID: 17289670.
- Moon AF, Garcia-Diaz M, Bebenek K, Davis BJ, Zhong X, Ramsden DA, Kunkel TA, Pedersen LC. Structural insight into the substrate specificity of DNA Polymerase mu. Nat Struct Mol Biol. 2007 Jan;14(1):45-53. Epub 2006 Dec 10. Erratum in: Nat Struct Mol Biol. 2007 Jul;14(7):680. PubMed PMID: 17159995.
- Povirk LF, Zhou RZ, Ramsden DA, Lees-Miller SP, Valerie K. Phosphorylation in the serine/threonine 2609-2647 cluster promotes but is not essential for DNA-dependent protein kinase-mediated nonhomologous end joining in human whole-cell extracts. Nucleic Acids Res. 2007;35(12):3869-78. Epub 2007 May 25. PubMed PMID: 17526517; PubMed Central PMCID: PMC1919499.
- Reddy YV, Perkins EJ, Ramsden DA. Genomic instability due to V(D)J recombination-associated transposition. Genes Dev. 2006 Jun 15;20(12):1575-82. PubMed PMID: 16778076; PubMed Central PMCID: PMC1482478.
- Nick McElhinny SA, Havener JM, Garcia-Diaz M, Juárez R, Bebenek K, Kee BL, Blanco L, Kunkel TA, Ramsden DA. A gradient of template dependence defines distinct biological roles for family X polymerases in nonhomologous end joining. Mol Cell. 2005 Aug 5;19(3):357-66. PubMed PMID: 16061182.
32-044 Lineberger Cancer Center
Campus Box 7295
Chapel Hill, NC 27599