Composite Active Site of an ABC ATPase

Junop, M.S.; Obmolova, Galina; Rausch, Kelly M.; Hsieh, Peggy; Yang, Wei

doi:10.1016/s1097-2765(01)00149-6

Cited by 234 publications

(163 citation statements)

References 44 publications

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“…Sliding of any more elaborate complex past a stiff hairpin extending about 70 Å from a 20-Å diameter helix would seem unlikely, virtually impossible if the complex included PCNA, whose central hole is 34 Å in diameter (27). Our data clearly do not falsify the hypothesis of Hsieh co-workers (9,10), that mismatch-activated searching complexes, containing hMutS␣, MutL␣, and perhaps other proteins, might remain at or near the mismatch and search for the nick through space.…”

Section: Fig 2 Mmr Substratescontrasting

confidence: 41%

“…In these cases, PCNA might play two roles even before DNA resynthesis: (i) tethering MutS␣ to homoduplex DNA via the known MutS␣⅐PCNA interactions, but releasing it when a mismatch is encountered, thus enhancing the efficiency of mismatch recognition, as suggested by other work (26), and (ii) allowing more elaborate recognition complexes, perhaps involving as well MutL␣, excision factors, and PCNA itself, to slide toward excision-initiation sites (25). We find here that purified hMutS␣ alone, apparently in contrast to bacterial MutS protein (9), is not retained on mismatched DNA blocked at both ends with hairpins in the presence of ATP. Thus, our experiments do not falsify any hypothesis in which ATP-dependent sliding of MutS␣ alone, either directly from a mismatch or after a cycle of MutL␣ recruitment and release by PCNA, searches for the excision-initiation signal, but do demand that the mode of any such sliding be loose enough to permit movement past the hairpin.…”

Section: Fig 2 Mmr Substratesmentioning

confidence: 53%

“…In one class of coupling models, mismatch-bound MutS/MSH dimers are postulated to bind ATP and then to move away along the DNA contour, searching for excision-initiation signals either by ATP hydrolysis-dependent translocation (7), or by ATP binding-dependent diffusional sliding, with hydrolysis occurring later (8). Or searching might be performed by more elaborate sliding complexes, to which other proteins have been recruited (25), Alternatively, MutS homolog proteins complexed with MutL homolog proteins and ATP are proposed to remain at or near mismatches, where they use DNA looping to search through space for excision initiation signals (9,10).…”

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confidence: 99%

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Mismatch Repair in Human Nuclear Extracts

Wang

Hays

2003

Journal of Biological Chemistry

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DNA mismatch repair (MMR) couples recognition of base mispairs by MSH2⅐MSH6 heterodimers to initiation, hundreds of nucleotides away, of nascent strand 3 -5 or 5 -3 excision through the mispair. Mismatchrecognition complexes have been hypothesized to move along DNA to excision-initiation signals, in eukaryotes, perhaps ends of nascent DNA, or to remain at mismatches and search through space for initiation signals. Subsequent MMR excision, whether simple processive digestion of the targeted strand or tracking of an excision complex, remains poorly understood. In human cell-free extracts, we analyzed correction of a mismatch in a 2.2-kilobase pair (kbp) circular plasmid containing a pre-existing excision-initiation nick for initiation, and measured MMR excision (in the absence of exogenous dNTPs) at specific locations. Excision specificities were ϳ100:1 for nicked versus continuous strands, 80:1 for mismatched versus homoduplex DNA, and 30:1 for shorter (0.3-kbp) versus longer (1.9-kbp) nick-mispair paths. To test models for recognition-excision coupling and excision progress, we inserted potential blockades, 20-bp hairpins, into nick-mispair paths, using a novel technique to first generate gapped plasmid. Continuous strand longer-path hairpins did not affect mismatch correction, but shorter-path hairpins reduced correction 4-fold, and both together eliminated it. Shorter-path hairpins had little effect on initiation of (3 -5 ) excision, measured 30 -60 nucleotides 5 to the nick, but blocked subsequent progress of excision to the mismatch; longer-path hairpins blocked the (lower level) 5 -3 excision to the mismatch. Thus, (a) MMR excision protein(s) cannot move past DNA hairpins. Hairpins at both ends of substrate-derived 0.5-kbp DNA fragments did not prevent ATP-induced dissociation of mismatch-bound human MSH2⅐MSH6, so recognition complexes at mismatches might provoke excision at nicks beyond hairpins, or loosely sliding MSH2⅐MSH6 dimers might move to the nicks.Evolutionarily conserved prokaryotic and eukaryotic mismatch-repair (MMR) 1 systems promote genomic stability by correcting DNA replication errors, antagonizing homeologous recombination between diverged DNA sequences, and responding to a variety of DNA lesions (for reviews, see Refs. 1-3). The Escherichia coli pathway, fully reconstituted from purified proteins, provides a mechanistic paradigm. Here, MutS homodimers bind to DNA mismatches and MutH proteins recognize hemi-methylated d(GATC) sequences, transitory consequences of a brief post-replication delay in GATC-adenine methylation of nascent DNA, to thus provide the two requisite specificity elements for correction. Interaction of MutL homodimers with MutS-mismatch complexes activates DNAstrand nicking of nascent DNA at unmethylated d(GATC) sites. DNA helicase II (UvrD protein) loads at the nick if and only if MutS, a DNA mismatch, and MutL are present; one or more steps in this chain of events require ATP hydrolysis. UvrD presumably separates strands to facilitate excision of the nicked strand towar...

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Section: Fig 2 Mmr Substratescontrasting

confidence: 41%

Section: Fig 2 Mmr Substratesmentioning

confidence: 53%

mentioning

confidence: 99%

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Mismatch Repair in Human Nuclear Extracts

Wang

Hays

2003

Journal of Biological Chemistry

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“…Both contain Walker A and B consensus sequences as well as the highly conserved 'LS-GGQ' signature sequence or C-motif. In DNA mismatch repair, the MutS ABC ATPase uses ATP binding to recognize and bind misrepaired DNA (Junop et al, 2001). In double-stranded break repair, Rad50 uses ATP to bind and bridge DNA double-strand breaks (Chen et al, 2001;Lobachev et al, 2002), and ABC transporters use the energy from ATP hydrolysis to transport substances across membranes (Hyde et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Molecular and Genetic Analysis of Condensin Function in Vertebrate Cells

Hudson

Ohta

Freisinger

et al. 2008

MBoC

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We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.

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“…One invokes movement of MutS or the MutL-MutS complex along the helix between the mismatch and the strand signal (11-13), a second posits mismatch recognition by MutS as a trigger for polymerization of a second protein along the helix between the two DNA sites (11), and the third attributes interaction of the two sites to a DNA-looping mechanism (14,15). Several laboratories have demonstrated that, when challenged with ATP, MutS homologs leave a mismatch by movement along the helix contour, and evidence for movement of the MutL-MutS homolog complex along DNA also is available (reviewed in refs.…”

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confidence: 99%

Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair

Pluciennik¹,

Modrich

2007

Proc. Natl. Acad. Sci. U.S.A.

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The hemimethylated d(GATC) sequence that directs Escherichia coli mismatch repair can reside on either side of a mismatch at a separation distance of 1,000 bp or more. Initiation of repair involves the mismatch-, MutS-, and MutL-dependent activation of MutH endonuclease, which incises the unmethylated strand at the d(GATC) sequence, with the ensuing strand break serving as the loading site for the appropriate 3 -to-5 or 5 -to-3 excision system. However, the mechanism responsible for the coordinated recognition of the mismatch and a hemimodified d(GATC) site is uncertain. We show that a protein roadblock (EcoRIE111Q, a hydrolytically defective form of EcoRI endonuclease) placed on the helix between the two DNA sites inhibits MutH activation by 70 -80% and that events that escape inhibition are attributable, at least in part, to diffusion of EcoRIE111Q away from its recognition site. We also demonstrate that a double-strand break located within the shorter path linking the mismatch and a d(GATC) site in a circular heteroduplex abolishes MutH activation, whereas a double-strand break within the longer path is without effect. These findings support the idea that initiation of mismatch repair involves signaling along the helix contour.DNA repair ͉ genetic stability ͉ signaling M ismatch repair is a conserved process that corrects biosynthetic errors and ensures the fidelity of homologous genetic recombination. Eleven activities have been implicated in Escherichia coli methyl-directed mismatch repair that has been reconstituted in a purified system. The reaction depends on MutS, MutL, MutH, DNA helicase II (MutU/UvrD), singlestranded DNA-binding protein, exonuclease I, exonuclease VII, exonuclease X, RecJ exonuclease, DNA polymerase III holoenzyme, and DNA ligase (1-4). The strand specificity necessary for replication error correction by this system is based on the transient absence of d(GATC) methylation in newly synthesized DNA (5).Repair is initiated via mismatch recognition by MutS (6), which recruits MutL to the heteroduplex in a mismatch-and ATP-dependent fashion (7,8). Assembly of the MutL-MutSheteroduplex complex is sufficient to activate MutH endonuclease, which incises the unmethylated strand of a hemimethylated d(GATC)-strand signal (9) that may reside either 3Ј or 5Ј to the mismatch at distances of as much as 1 kb or more (2). MutS and MutL also activate DNA helicase II, which is loaded at the MutH strand break in an orientation-dependent manner, so that helix unwinding proceeds toward the mismatch (10). That portion of the incised strand displaced in this manner is degraded by a single-stranded exonuclease, resulting in mismatch removal. When the MutH nick is 3Ј to the mismatch, the single-stranded hydrolytic requirement can be met by exonuclease I, exonuclease VII, or exonuclease X, and, when the nick is 5Ј to the mispair, either exonuclease VII or RecJ will suffice in this regard (2-4). The mechanism responsible for action at a distance during mismatch repair is poorly understood, but the bidirectional ...

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Composite Active Site of an ABC ATPase

Cited by 234 publications

References 44 publications

Mismatch Repair in Human Nuclear Extracts

Mismatch Repair in Human Nuclear Extracts

Molecular and Genetic Analysis of Condensin Function in Vertebrate Cells

Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair

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