Differential Specificities and Simultaneous Occupancy of Human MutSα Nucleotide Binding Sites

Abstract

Mismatch repair corrects DNA biosynthetic errors, ensures the fidelity of genetic recombination, and participates in the cellular response to certain types of DNA damage (1-6). Assembly of MutS and MutL homologues at a mismatch or at a DNA lesion is presumed to be an early event in each of these genetic stabilization functions. In human cells the MutS homologue MSH2 forms heterodimers with MSH6 (7-10) or MSH3 (10, 11), and these activities are responsible for mismatch recognition. The MutS␤ heterodimer (MSH2⅐MSH3) recognizes small insertion/deletion mispairs (9, 10, 12), but MutS␣ (MSH2⅐MSH6) recognizes and supports the repair of both basebase mismatches and insertion/deletion heterologies (7, 12, 13).In the human cell lines that have been examined, the majority of the MSH2 is associated with MSH6 (11,12,14), and the MutS␣ complex is present in a 6-to 10-fold excess over MutS␤.In addition to their mismatch recognition function, MutS homologues contain a highly conserved ATP hydrolytic center near the carboxyl terminus (15). The integrity of this site is required for function in mismatch repair (15-17), and there is extensive evidence indicating that adenine nucleotides modulate the interaction between MutS homologues and DNA. ATP and nonhydrolyzable ATP analogues reduce MutS homologue affinity for a mispair (7,8,(17)(18)(19)(20)(21)(22)(23), and nucleotide challenge of preformed protein⅐heteroduplex complexes provokes MutS homologue release from a mismatch (23-28).Several models for ATPase function in MutS homologue action have been suggested. Two of these invoke ATP-dependent movement of the protein along the helix, which is postulated to link mismatch recognition to activation of downstream events at the strand signal that directs repair (24 -26). Electron microscopic visualization of bacterial MutS⅐heteroduplex complexes has demonstrated the mismatch-and ATP-dependent extrusion of a DNA loop by the bacterial protein (24). Because nonhydrolyzable ATP analogues do not support loop extrusion and because their addition terminates ongoing loop growth, this reaction has been attributed to directional translocation along the helix in a reaction dependent on ATP hydrolysis by the DNA-bound protein. The use of streptavidin end-blocked linear DNAs has also suggested that MutS and MutS␣ leave a mismatch in an ATP-dependent manner, observations that have led to the suggestion that the protein may form a mobile clamp about the helix (22,25,26,29). Although ATP⅐Mg 2ϩ supports the formation of stable complexes on end-blocked heteroduplexes, AMPPNP⅐Mg 2ϩ , 1 ATP␥S⅐Mg 2ϩ , or ATP (no Mg 2ϩ ) do not, suggesting that formation of such intermediates may depend on ATP hydrolysis by heteroduplex-bound MutS␣ (25). The interaction with end-blocked DNA of a mutant form of MutS␣, which supports ATP binding but not hydrolysis, has led to a similar conclusion (27).A mechanism for MutS/MutS␣ movement along the helix that is independent of ATP hydrolysis by DNA-bound protein has also been proposed (26,29). This molecular switch model posi...

Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D

“…Quantitative assessment by fluorescence anisotropy revealed high affinity binding to G:T by all the proteins in the absence of nucleotides or in the presence of ADP ( Table 1). As expected, wild type hMutS␣ affinity for G:T was substantially reduced in the presence of ATP, in agreement with a previous report (40). In contrast, mismatch binding by the G674A and T1219D mutants was insensitive to ATP (Table 1).…”

Asymmetric ATP Binding and Hydrolysis Activity of the Thermus aquaticus MutS Dimer Is Key to Modulation of Its Interactions with Mismatched DNA

“…For example, crystal structures of both T. aquaticus and E. coli MutS proteins show that a phenylalanine from only one subunit stacks against an unpaired or mispaired base in the duplex (14,15), and the E. coli MutS structure shows an ADP molecule bound to only one subunit (15). Consistent with this structural data, the Modrich research group detected only one ADP, AMPPNP, or ATPγS molecule binding per E. coli MutS, as well as human Msh2-Msh6, dimer (29,47). At the same time, our study of S. cerevisiae Msh2-Msh6 detected only one ATP or one ADP binding per heterodimer, and we also demonstrated that two ATPγS molecules bind the dimer with differing affinities (3-5-fold difference, ref 31).…”

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…In addition, the two nucleotide binding sites are asymmetric with binding constants in the 1 -20 μM range. A number of biochemical studies has established that the two ATP binding sites are nonidentical with MSH6 (and presumably its bacterial counterpart) having a high affinity for ATP and MSH2 having a higher affinity for ADP Hingorani, 2003, 2004;Antony et al, 2006;Bjornson and Modrich, 2003;Martik et al, 2004). Seemingly identical mutations in the ATP binding sites of either MSH2 or MSH6 can have different effects on MMR in vivo and DNA binding and ATP hydrolysis in vitro supporting the notion that nucleotide binding/hydrolysis plays a central role in regulating the activities of MutS proteins (Jacobs-Palmer and Hingorani, 2007).…”