Dana Farber Cancer Institute
Jimmy Fund Building, Room 517
44 Binney Street
Boston, MA 02115
Down regulating DNA repair- phosphatases and micro RNAs
Activation of DNA repair pathways have been well studied for decades with very limited research on down regulation. Most importantly, the physiological relevance of down modulating DNA repair is still unclear. We have focused on two aspects of down regulation, one is dephosphorylation of repair proteins via phosphatases, and second is decreased expression of repair factors via micro RNAs (miRNAs).
In response to double stranded DNA breaks (DSBs), ~700 mouse and human proteins including factors involved in DNA replication and repair, apoptosis, and/or cell cycle progression are phosphorylated by the (PI3-like) family of kinases. Although the role of kinases in the DSB reponse is very well established, phosphatases have only recently come into the spotlight. In the past few years we have identified a role for the PP2A-like phosphatases (PP2AC and PP4C) in the DSB response and observed that these phosphatases dephosphorylate a key DNA repair protein, the histone variant, H2AX (2005 Molecular Cell, 2008 Molecular Cell). Very recent studies investigating the DSB-induced dynamics of phosphorylation show that over one-third of the captured phospho-peptides are de-phosphorylated within minutes of DNA damage. This suggests that phosphatases not only play a role in counter-acting the DSB-induced phosphorylation later in the damage response, but they play a primary role in initiating the repair process. Importantly, this is consistent with our recent study (Nature Structural & Molecular Biology, 2010) showing that human protein phosphatase 4 (PP4) dephosphorylates the essential replication protein A (RPA) on the RPA2 subunit, immediately after DNA damage, and this dephosphorylation event is critical for efficient repair of DSBs. We are using a combination of sophisticated mass spectrometry and biochemical tools to systematically identify phosphatases involved in the DSB response. Most of the DNA repair factors (including H2AX and RPA) have critical roles V(D)J recombination and class-switch recombination that occur during lymphocyte development, we plan to study the impact of phosphatases on these recombination events.
miRNAs are small non-coding RNAs that typically dampen gene expression and are mis-expressed in many cancer cells. Although miRNAs are being widely studied in different cellular process, to date there is limited information regarding their role in DNA repair. Using a model of in vitro hematopoietic cell differentiation and miRNA expression analysis, we recently identified miRNAs that target DSB repair factors (Nature Structural & Molecular Biology, 2009). We showed that miR-24 downregulates the expression of a key DSB repair protein, H2AX, impedes DSB repair, and sensitizes terminally differentiated blood cells, including cells involved in the innate immune system, to DNA damaging agents. This is the first example of a miRNA regulating the efficiency of DNA repair. We have done multiple screens to identify miRNAs impacting DNA repair, and find that the expression of these miRNAs correlate with therapeutic response of cancer patients, underlining their clinical relevance. We adapted a biochemical strategy of co-immunoprecipitating miRNA/mRNA interactions to identify physiologically relevant targets of miRNAs. Conversely, we are devising new strategies to identify miRNAs targeting specific DNA repair factors. A role for miRNAs in DNA repair will be extremely pertinent for understanding how they impact DNA recombination in lymphocytes.
D Chowdhury, MC Keogh, H Ishii, CL Peterson, S Buratowski, J Lieberman, "γ-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double strand break repair," Mol Cell 2005 Dec 9;20(5):801-9.
D Chowdhury*, X Xu*, X Zhong, F Ahmed, J Zhong, J Liao, D M Dykxhoorn, D M Weinstock, G P Pfeifer, and J Lieberman* “A PP4-phosphatase complex dephosphorylates γ-H2AX generated during DNA replication,” Mol Cell 2008 July 11;31, 1-14 * Joint Corresponding
A Lal, Y Pan, F Navarro, DM. Dykxhoorn, L Moreau, J Lieberman, D Chowdhury, “miR-24-mediated down-regulation of H2AX suppresses DNA repair in terminally differentiated cells”, Nat Struct Mol Biol. 2009 May;16(5):492-8
D Lee, Y Pan, S Kanner, P Sung, J A. Borowiec and D Chowdhury. “A PP4-phosphatase complex dephosphorylates RPA2 facilitating homologous recombination-mediated DNA repair.” Nature Structural & Molecular Biology 2010 Mar;17(3):365
Last Update: 1/6/2014