DNA Chain Length Dependence of Formation and Dynamics of hMutSα·hMutLα·Heteroduplex Complexes

Abstract

Formation of a ternary complex between human MutS␣, MutL␣, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutL␣⅐hMutS␣⅐heteroduplex complex requires a mismatch and ATP hydrolysis, and depends on DNA chain length. Ternary complex formation was supported by a 200-base pair G-T heteroduplex, a 100-base pair substrate was somewhat less effective, and a 41-base pair heteroduplex was inactive. As judged by surface plasmon resonance spectroscopy, ternary complexes produced with the 200-base pair G-T DNA contained ϳ0.8 mol of hMutL␣/mol of heteroduplex-bound hMutS␣. Although the steady-state levels of the hMutL␣⅐hMutS␣⅐ heteroduplex were substantial, this complex was found to turn over, as judged by surface plasmon resonance spectroscopy and electrophoretic gel shift analysis. With the former method, the majority of the complexes dissociated rapidly upon termination of protein flow, and dissociation occurred in the latter case upon challenge with competitor DNA. However, ternary complex dissociation as monitored by gel shift assay was prevented if both ends of the heteroduplex were physically blocked with streptavidin⅐biotin complexes. This observation suggests that, like hMutS␣, the hMutL␣⅐hMutS␣ complex can migrate along the helix contour to dissociate at DNA ends.The MutS and MutL homologs, which are required for the initiation of mismatch repair, have been implicated in the correction of DNA biosynthetic errors, the transcription-coupled repair of DNA damage, and the fidelity of genetic recombination (1-6). In mammalian cells, MutS␣ (MSH2⅐MSH6 heterodimer), MutS␤ (MSH2⅐MSH3 heterodimer), and MutL␣ (MLH1⅐PMS2 heterodimer) are also thought to function as lesion sensors for certain types of DNA damage that kill by activating apoptosis (2, 6, 7).Repair in the Escherichia coli system is initiated by the binding of MutS to a mismatch (8 -10). Formation of a MutL⅐MutS⅐heteroduplex complex has been demonstrated by DNase I footprint analysis (11), electron microscopy (12), and surface plasmon resonance spectroscopy (SPRS) 1 (13), with assembly of this ternary complex being ATP-dependent. Several lines of evidence indicate that assembly of the ternary complex is required for subsequent steps in mismatch repair. Both MutS and MutL are required for the mismatch-dependent activation of the d(GATC) endonuclease activity of MutH, which cleaves the unmethylated strand of a hemimethylated d(GATC) site, with the ensuing strand break serving to direct repair to the unmethylated DNA strand (14). MutS and MutL are also required for the mismatch-dependent activation of DNA helicase II, which enters the helix at the strand break and initiates the excision step of repair (15). Formation of a MutL␣⅐MutS␣⅐heteroduplex complex has been demonstrated by electrophoretic gel shift analysis with yeast mismatch repair proteins using synthetic heteroduplexes of ϳ50 base pairs (bp) in size (16, 17). However, using gel shift methods and surface plasmon reson...

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…In addition, DNasel footprinting studies reveal that whereas MutS protects approximately one turn of the DNA helix to either side of the mismatch consistent with the crystal structures, the MutS-MutL interaction in the presence of ATP yields a very large DNasel footprint indicative of multiple proteins bound to the DNA (Grilley et al, 1989;Selmane et al, 2003). Formation of a ternary complex involving either the E. coli or human MMR proteins also requires DNA heteroduplexes in excess of 60 bp reflecting multiple binding events in vitro (Blackwell et al, 2001;Plotz et al, 2002;Schofield et al, 2001b). Whether this is an artefact of in vitro conditions or reflects multiple loadings of MMR proteins, either multiple loading of diffusing clamps or cooperative binding of a protein filament, is not definitively established.…”

“…ATP-induced dissociation of yMsh2-Msh6 from a mismatch occurs preferentially from DNA ends though direct dissociation from the DNA is also observed Mendillo et al, 2005). Finally, although MutS and MutL (and their eukaryotic homologues) have been observed to form quasi-stable complexes at sites of mismatches in vitro (Blackwell et al, 2001;Räschle et al, 2002;Schofield et al, 2001 b), yeast and human complexes have also been observed to move along the DNA contour in the presence of ATP in surface plasmon resonance experiments (Blackwell et al, 2001;Mendillo et al, 2005).…”

PHF11 promotes DSB resection, ATR signaling, and HR

“…Beads were washed three to five times in the same buffer. Five micrograms to 10 µg of GST-hPHF11 or GST alone were incubated with various amounts of RPA or EXO1 protein purified as described previously (Henricksen et al 1994;Blackwell et al 2001) in binding buffer (50 mM HEPES-KOH at pH 7.9, 150 mM NaCl, 1.5 mM MgCl 2 , 0.5% [v/v] NP40, 1 mM DTT, 1 mM PMSF, Roche protease inhibitor mix) for 4-6 h at 4°C. Beads were collected at 500g and washed three times with binding buffer, and bound protein was eluted in 2× Laemmli buffer and analyzed by immunoblotting.…”

A conserved MutS homolog connector domain interface interacts with MutL homologs

“…Mispairs in DNA are recognized by the MutS homodimer in Escherichia coli or by one of two heterodimers of MutS homologs, Msh2-Msh6 or Msh2-Msh3, in eukaryotes (2,10,11). This complex then recruits the MutL homodimer in E. coli or, in eukaryotes, one of two MutL heterodimeric complexes, Mlh1-Pms1 or Mlh1-Mlh3, in an ATP-dependent manner (12)(13)(14)(15)(16). In E. coli, MutS-MutL-DNA ternary complex stimulates the endonucleolytic activity of MutH, which makes single-strand breaks in the unmethylated DNA strand at transiently hemimethylated GATC sites and thus distinguishes the unmethylated daughter DNA strand from the methylated parental DNA strand during and after DNA replication (17)(18)(19).…”