Monday, March 22, 2010

DNA Repair 5: The final chapter

Another method of DSB repair is via non-homologous end joining (NHEJ), which seems significantly riskier for the cell because it involves the ligation of two ends of DNA.  Nonetheless, it appears that this is the preferred mode of double-stranded breaks and is not random.  Hypotheses have been considered that the proteins involved in NHEJ are very abundant in the cell, and when DSB occur, they are immediately available to attach to the DNA and ligate it back together.  This process is performed by Ku heterodimers, which are proteins that bind directly to the ends at the DSBs.  Ku proteins then recruit DNA-PKcs (DNA protein kinases), which, in turn, recruit Artemis endonuclease to open the ends and remove any hairpin structures that may have developed.  The ends of the DNA are then slightly chewed away, polymerized, phosphorylated, and ligated together.  The action of XRCC4, another protein that complexes during NHEJ, and Ku is used to recruit DNA ligase IV.  The final product of this process is a DNA strand that is very similar to the original DNA, but it is not exact:  NHEJ does result in mutated DNA sequences.

An excellent review on DSBs and their repair can be found here.

Double-stranded breaks sound like a terrible thing to have happen to a cell.  After all, when DNA breaks and is repaired, there is a high chance that the mutation that arises will be harmful to the cell.  Nonetheless, double-stranded breaks aren’t detrimental to the cell, and they are even required for certain normal cellular processes.  For example, in the development of immunoglobulins, lymphocytes’ genomes are rearranged and parts are removed in a process termed VDJ recombination.  This allows for the lymphocytes to increase diversity in the coding regions of the immunoglobulin genes and helps to protect us from disease.  To begin VDJ recombination, site-specific endonucleases RAG-1 and RAG-2 induce DSBs and repair these breaks via NHEJ.

As mentioned above, Ku proteins bind directly to DSBs when they occur, but how do they know when a DSB occurs?  The detection of these breaks is performed by the MRN complex composed of Mre1, Nbs1, and Rad50.  Mre11 acts as an endonuclease, Rad50 as an ATPase, and Nbs1 as a scaffold.  These proteins, once in complex, hold the DNA together and recruit other proteins, resulting in several signaling cascades.  These signaling cascades have been studied in detail using ionizing radiation induced foci (IRIF) in vitro.  The data that has been gathered from these studies shows that the MRN complex recruits ATM, a PI3K-family kinase, that becomes active during DSB formation.  ATM then phosphorylates H2AX, an H2A variant, to form γH2AX.  γH2AX acts as a scaffold to recruit more ATM and many other proteins, such as chromatin remodeling complexes, to the site of the lesion.  The cell cycle is arrested via phosphorylation of Chk2 by ATM (or phosphorylation of Chk1 by ATR – ATM-related kinase).  Chk1 and Chk2 phosphorylate CDC25 phosphatases and inactivate them, which prevents the activation of cyclin-dependent kinases.  Additionally, ATM can induce phosphorylation and activation of p53, leading to G1/S arrest.  Tip60 is another protein involved in DNA repair and is involved in acetylating ATM to activate it.  ATM and Tip60 exist in a constitutive complex that is only activated on DNA damage signaling.  MCD1 acts as a scaffold that binds phosphorylated H2AX and docks ATM/Tip60.

DSB detection simplified:
  1. The MRN complex recognizes and binds the free ends of the DNA
  2. ATM is recruited to the foci
  3. ATM phosphorylates H2AX, Chk2 and p53
  4. Chk2 phosphorylates CDC25
  5. Cell cycle is arrested and chromatin opens for DNA repair

Several diseases are associated with defects in the DSB response pathway.  Among them are ataxia-telangiectasia, resulting from mutations in ATM.  Several mutations in ATM will result in the A-T phenotype, and approximately 1% of the population carries one mutated allele.  Those that suffer from this syndrome show immunodeficiency and increased cancer rates, and they have a life expectancy of 17-23 years.  In addition to A-T, Nijimegen breakage syndrome results from mutations in Nbs1, and A-T like disorder (ATLD) results from Mre11 mutations.  Breast cancer may also have connections to DSB repair, as BRCA1 is associated with the BASC complex, which is involved in this and many other pathways for DNA repair.  BRCA1 acts as an ATM substrate and localizes to IRIFs, possibly acting as a scaffold.

More information about ataxia-telangiectasia can be found here.
On NBS can be found here.
A paper about ATLD: here.

Identification of genes involved in DNA repair
A number fo methods have been developed that specifically scan organisms in order to detect defects in DNA repair.  Isolation of the affected gene and protein then allows for recognition of specific DNA repair proteins.  Specifically, one can look at the frequency of mutations (see Ames test, for example) as well as elevated sensitivity to ultra-violet or ionizing radiation.  Cell cycle arrest (cell division defects) and apoptosis are common phenotypes associated with defective enzymes as well.

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