Friday, March 19, 2010

DNA Repair 4

A further method of repairing small lesions in the DNA is via mismatch repair (MMR), which fixes errors in replication and heteroduplexes from recombination intermediates.  The process involves the Mut proteins in prokaryotes.  MutS bound to ATP recognizes and binds the unpaired base to activate the mismatch repair enzymes.  MutH is involved in determining which DNA strand is methylated, indicating the parent strand (the correct copy of the DNA that should be used to repolymerize the incorrect copy).  MutL facilitates in assembling the MMR complex, which consists of helicases and exonucleases.  Repair is then done by DNA polymerase and ligase, and methylation of the newly copied (and unmutated) strand is then performed.  MutL and MutS homologues are found in both yeast and humans, with varying roles and functioning as heterodimers.  Additionally, in eukaryotic MMR, the ability of the enzymes to distinguish between parent and daughter strands is not as clear.  Hypotheses have emerged that consider interaction with PCNA and identification of nicked strands (on the nascent DNA) as mechanisms that may assist in determining which strand needs to be repaired.
 
One consequence of an impaired mismatch repair system is microsatellite instability, a variation in tandem repeat sequences.  In cells both with and without a functioning MMR system, changes in the number of repeats is commonly observed as the DNA is being replicated.  This variation is due to slippage of DNA polymerase as it runs through repeated sequences, as if it has gotten lost along the way.  Usually, the microsatellite instability is held in check by MMR enzymes that are able to recognize the bubble that forms in the DNA.  However, in those individuals with mutations, there can be a vast array of repeat numbers in these microsatellites, up to 3.3x10-3 mutations per cell per generation.   

The most catastrophic event that can occur in a cell is double-stranded breaks (DSBs) in the DNA, which can be caused by radiation and mutagens, but it can also result from DNA replication and physiological processes (harmful metabolites that are not controlled by the cell properly).  Two methods have evolved to prevent these DSBs from having a negative impact on the cell, which we will consider now.

The first method of DSB repair is by homologous recombination.  (Details about this pathway can be found in additional posts about recombination.)  There are two methods for recombination: synthesis-dependent strand annealing and single-strand annealing.  In synthesis-dependent strand annealing homologous recombination, strand invasion of the undamaged DNA (sister chromosome) by the DNA that contains the double-stranded break.  New DNA is synthesized from the correct copy and ligated to remove the DSB.  In contrast to synthesis-dependent annealing, single-strand annealing relies on an uneven breakage in the DNA.  Several bases on each side of the DNA strand are removed, and the staggered ends are annealed to create fixed DNA.  This method of DSB repair is especially common in stretches of repeated-sequence DNA.

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