MutS, MutL, and DNA helicase II are required for the mismatch-provoked excision step that occurs during Escherichia coli methyl-directed mismatch repair. In this study MutL is shown to enhance the unwinding activity of DNA helicase II more than 10-fold on a conventional helicase substrate in which a 35-residue oligonucleotide is annealed to a M13 circular single-stranded phage DNA under conditions where the two proteins are present at approximately molar stoichiometry with respect to the substrate. MutS-and MutL-dependent activation of DNA helicase II has also been demonstrated with a model substrate in which a 138-residue oligonucleotide was hybridized to a 138-nucleotide gap in an otherwise duplex 7,100-base pair circular DNA. Displacement of the oligonucleotide requires MutS, MutL, DNA helicase II, and ATP and is dependent on the presence of a mismatch within the hybrid region. Although DNA helicase II and Rep helicase share substantial sequence homology and features of mechanism, Rep helicase is inactive in this reaction.Escherichia coli methyl-directed mismatch repair initiates via the mismatch-provoked incision of the unmethylated strand at a hemimethylated d(GATC) sequence in a reaction that involves the MutS-and MutL-dependent activation of the MutH d(GATC) endonuclease activity (1). The single-strand break thus produced may occur either 3Ј or 5Ј to the mismatch on the unmethylated strand and directs the exonucleolytic excision of that portion of the unmodified strand spanning the incised d(GATC) sequence and the mispair (2, 3). Excision requires MutS, MutL, DNA helicase II, and an appropriate exonuclease. When the strand break that directs repair occurs 5Ј to the mismatch, excision requires RecJ exonuclease or exonuclease VII (3, 4), both of which support 5Ј 3 3Ј hydrolysis (5, 6). For repair directed by a strand break 3Ј to the mismatch, the 3Ј 3 5Ј hydrolytic activity of exonuclease I (7) is sufficient to meet the exonuclease requirement (2). 1Since helicase II is required for excision from either side of the mismatch and because each of these exonucleases is specific for single-stranded DNA (5-7), the action of helicase II presumably serves to unwind the incised strand so as to render it exonuclease sensitive. According to this interpretation, the exonuclease functions in excision are secondary to those of DNA helicase II. We have therefore sought partial reactions in which MutS, MutL, and a mismatch might enhance the activity of helicase II. We show here that MutL stimulates helicase II activity on a conventional substrate and that helicase activity on incised duplex DNA is enhanced by MutS and MutL in a mismatch-dependent manner. The accompanying paper (8) demonstrates that MutS, MutL, and mismatch-dependent entry of helicase II into an incised heteroduplex occurs at the strand break with helicase entry biased so that translocation occurs toward the mispair. EXPERIMENTAL PROCEDURESProteins, DNA, and Nucleotides-MutS (9) and DNA helicase II (10) were purified as described. Rep helicase (11) was kind...

Escherichia coli MutS, MutL, and DNA helicase II are sufficient to initiate mismatch-dependent unwinding of an incised heteroduplex (Yamaguchi, M., Dao, V., and Modrich, P. (1998) J. Biol. Chem., 273, 9197-9201). We have studied unwinding of 6.4-kilobase circular G-T heteroduplexes that contain a single-strand incision, 808 base pairs 5 to the mismatch or 1023 base pairs 3 to the mispair as viewed along the shorter path between the two DNA sites. Unwinding of both substrates in the presence of MutS, MutL, DNA helicase II, and singlestranded DNA binding protein was mismatch-dependent and initiated at the single-strand break. Although unwinding occurred in both directions from the strand break, it was biased toward the shorter path linking the strand break and the mispair. MutS and MutL are thus sufficient to coordinate mismatch recognition to the orientation-dependent activation of helicase II unwinding at a single-strand break located a kilobase from the mispair.The strand specificity necessary for correction of DNA biosynthetic errors by the Escherichia coli mismatch repair system is provided by the transient absence of adenine modification of d(GATC) sequences within newly synthesized DNA (1). Repair is initiated by binding of a MutS homodimer to a mismatch followed by addition of MutL to this complex (2-4). Assembly of this ternary complex activates a MutH-associated endonuclease that cleaves the unmethylated strand at a hemimethylated d(GATC) sequence within newly replicated DNA (5). The single-strand break introduced by MutH, which may occur either 3Ј or 5Ј to the mismatch on the unmethylated strand, directs the excision of that portion of the unmodified strand spanning the d(GATC) sequence and the mispair (6, 7). Excision requires MutS, MutL, DNA helicase II (also called MutU), and depending on the strand break to mismatch orientation, a 3Ј 3 5Ј or 5Ј 3 3Ј single-strand exonuclease (6,8).The accompanying manuscript (9) demonstrates that MutS and MutL greatly enhance the activity of DNA helicase II on incised heteroduplex DNA. In this paper we have used KMnO 4 to determine the site of initiation of mismatch-dependent helix unwinding in DNA substrates containing a site-and strandspecific, single-strand break. Permanganate preferentially attacks single-stranded DNA where it oxidizes the 5,6 double bond of thymine and methylcytosine and reacts to a lesser degree with other bases (10, 11). This single-strand selective reagent has been used previously to detect helix opening associated with promoter melting by bacterial and eukaryotic RNA polymerases (12,13). Using this approach we show that MutS-, MutL-, and helicase II-dependent unwinding of an incised heteroduplex initiates at the strand break, with the direction of unwinding being biased toward the shorter path between the strand break and the mismatch in a circular heteroduplex. EXPERIMENTAL PROCEDURESProteins and DNA-E. coli MutS (14), MutL (3), and MutH (15) were purified as described previously. DNA helicase II was isolated from an overproducing strain a...

The efficiency of translation depends on correct tRNA-ribosome interactions.

Two single-stranded DNA heptadecamers corresponding to the yeast tRNA(Phe) anticodon stem-loop were synthesized, and the solution structures of the oligonucleotides, d(CCAGACTGAAGATCTGG) and d(CCAGACTGAAGAU-m5C-UGG), were investigated using spectroscopic methods. The second, or modified, base sequence differs from that of DNA by RNA-like modifications at three positions; dT residues were replaced at positions 13 and 15 with dU, and the dC at position 14 with d(m5C), corresponding to positions where these nucleosides occur in tRNA(Phe). Both oligonucleotides form intramolecular structures at pH 7 in the absence of Mg2+ and undergo monophasic thermal denaturation transitions (Tm = 47 degrees C). However, in the presence of 10 mM Mg2+, the modified DNa adopted a structure that exhibited a biphasic "melting" transition (Tm values of 23 and 52 degrees C) whereas the unmodified DNA structure exhibited a monophasic denaturation (Tm = 52 degrees C). The low-temperature, Mg(2+)-dependent structural transition of the modified DNA was also detected using circular dichroism (CD) spectroscopy. No such transition was exhibited by the unmodified DNA. This transition, unique to the modified DNA, was dependent on divalent cations and occurred most efficiently with Mg2+; however, Ca2+ also stabilized the alternative conformation at low temperature. NMR studies showed that the predominant structure of the modified DNA in sodium phosphate (pH 7) buffer in the absence of Mg2+ was a hairpin containing a 7-nucleotide loop and a stem composed of 3 stable base pairs. In the Mg(2+)-stabilized conformation, the loop became a two-base turn due to the formation of two additional base pairs across the loop.(ABSTRACT TRUNCATED AT 250 WORDS)

The tDNA(Phe)AC, d(CCAGACTGAAGAU13m5C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+)-induced structural transition that was not exhibited by an unmodified tDNA(Phe)AC d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., & Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(Phe)AC molecules having m5C14, tDNA(Phe)AC d(U13m5C14U15), d(U13m5C14T15), and d(T13,5C14U15), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(Phe)AC, d(T13C14T15), d(U13C14U15), and d(A7;U13m5C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+)-induced structural transitions and only monophasic thermal transitions (Tm of approximately 52 degrees C). The tDNA(Phe)AC d(U13m5C14U15) had a single, strong Mg2+ binding site with a Kd of 1.09 x 10(-6) M and a delta G of -7.75 kcal/mol associated with the Mg(2+)-induced structural transition. In thermal denaturation of tDNA(Phe)AC d(U13m5C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe).(ABSTRACT TRUNCATED AT 250 WORDS)

Cross reactivity of patient lupus autoantibodies to the small nuclear ribonucleoprotein particles of many different types of animals is well documented. The aim of our research was to determine if any level of cross reactivity existed between proteins of common dietary plants and anti-Sm autoantibodies of lupus patient's sera, as has been found for scleroderma patient sera (Agris et al., Exptl. Cell Res. 189, 276-279, 1990). Protein extracts from soy bean, corn, spinach, and carrot were analyzed. At least one protein (molecular weight approximately 28,000 daltons) common to all the above protein extracts was recognized by most of the anti-Sm sera tested. Affinity purified antibody eluted from the 28 kilodalton plant protein specifically recognized the Sm proteins of a HeLa cell protein extract. Recognition of the 28 kilodalton dietary plant protein was found to be unique to anti-Sm lupus sera.

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