Multiple DNA repair mechanisms and alkylator resistance in the human medulloblastoma cell line D-283 Med (4-HCR).

PURPOSE: We have previously reported preferential repair of DNA interstrand crosslinks in the 4-hydroperoxycyclophosphamide-resistant human medulloblastoma cell line D-283 Med (4-HCR). We now report further studies that explored the potential mechanisms underlying this repair. METHODS: Limiting dilution assays and Western, Southern, and Northern blots were used to compare specific differences between D-283 Med (4-HCR) and its parental line D-283 Med. RESULTS: D-283 Med (4-HCR) was cross-resistant to melphalan and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with O6-alkylguanine-DNA alkyltransferase (AGT) levels of 466+/-164 fmol/mg protein; AGT levels in the parental line, D-283 Med, were 76+/-96 fmol/mg. The increase in AGT activity was not a result of gene amplification. Depleting AGT with O6-benzylguanine partially restored sensitivity to BCNU. Both cell lines were deficient in the human mismatch protein MutLalpha. ERCC4 mRNA and poly(ADP-ribose) polymerase levels were similar in both cell lines, and ERCC1 mRNA levels were 2- to 2.5-fold lower in D-283 Med (4-HCR). Topoisomerase I levels were 2- to 2.5-fold higher in D-283 Med compared with D-283 Med (4-HCR). CONCLUSION: These results, while illustrating the multiple differences between D-283 Med and D-283 Med (4-HCR), do not explain the enhanced DNA interstrand crosslink repair seen in D-283 Med (4-HCR).

Kinetics of Escherichia coli helicase II-catalyzed unwinding of fully duplex and nicked circular DNA.

Escherichia coli helicase II (UvrD) protein can initiate unwinding of duplex DNA at blunt ends or nicks, although these reactions require excess protein. We have undertaken kinetic studies of these reactions in order to probe the mechanism of initiation of unwinding. DNA unwinding was monitored directly by using agarose gel electrophoresis and indirectly through the rate of ATP hydrolysis by helicase II in the presence of an ATP-regenerating system. In the presence of fully duplex DNA and excess helicase II, the rate of ATP hydrolysis displays a distinct lag phase before the final steady-state rate of hydrolysis is reached. This reflects the fact that ATP hydrolysis under these conditions results from helicase II binding to the ssDNA products of the unwinding reaction, rather than from an intrinsic duplex DNA-dependent ATPase activity. Unwinding of short blunt-ended duplex DNA (341 and 849 base pairs) occurs in an "all-or-none" reaction, indicating that initiation of unwinding by helicase II is rate-limiting. We propose a minimal mechanism for the initiation of DNA unwinding by helicase II which includes a binding step followed by the rate-limiting formation of an initiation complex, possibly involving protein dimerization, and we have determined the phenomenological kinetic parameters describing this mechanism. Unwinding of a series of DNA substrates containing different initiation sites (e.g., blunt ends, internal nicks, and four-nucleotide 3' vs 5' ssDNA flanking regions) indicates that the rate of initiation is slowest at nicks and, surprisingly, at ends possessing a four-nucleotide 3' ssDNA flanking region.

Overexpression, purification, DNA binding, and dimerization of the Escherichia coli uvrD gene product (helicase II).

We have subcloned the Escherichia coli uvrD gene under control of the inducible phage lambda PL promoter and report a procedure for the large-scale purification of helicase II protein. Yields of approximately 60 mg of > 99% pure helicase II protein, free of detectable nuclease activity, are obtained starting from 250 g of induced E. coli cells containing the overexpression plasmid. Overproduction of helicase II protein at these levels is lethal in E. coli. The extinction coefficient of helicase II protein was determined to be epsilon 280 = 1.06 (+/- 0.05) x 10(5) M-1 (monomer) cm-1 [20 mM Tris-HCl (pH 8.3 at 25 degrees C), 0.2 M NaCl, and 20% (v/v) glycerol, 25 degrees C]. We also present a preliminary characterization of the dimerization and DNA binding properties of helicase II and a systematic examination of its solubility properties. The apparent site size of a helicase II monomer on ss-DNA is 10 +/- 2 nucleotides as determined by quenching of the intrinsic tryptophan fluorescence of the protein upon binding poly(dT). In the absence of DNA, helicase II protein can self-assemble to form at least a dimeric species at concentrations > 0.25 microM (monomer) and exists in a monomer-dimer equilibrium under a variety of solution conditions. However, upon binding short oligodeoxynucleotides, the dimeric form of helicase II is stabilized, and dimerization stimulates the ss-DNA-dependent ATPase activity, suggesting that the dimer is functionally important. On the basis of these observations and similarities between helicase II and the E. coli Rep helicase, which appears to function as a dimer [Chao, K., & Lohman, T. (1991) J. Mol. Biol. 221, 1165-1181], we suggest that the active form of helicase II may also be a dimer or larger oligomer.

Escherichia coli helicase II (UvrD) protein initiates DNA unwinding at nicks and blunt ends.

The Escherichia coli uvrD gene product, helicase II, is required for both methyl-directed mismatch and uvrABC excision repair and is believed to function by unwinding duplex DNA. Initiation of unwinding may occur specifically at either a mismatch or a nick, although no direct evidence for this has previously been reported. It has recently been shown that helicase II can unwind fully duplex linear and nicked circular DNA with lengths of at least approximately 2700 base pairs in vitro; hence, a flanking region of single-stranded DNA is not required to initiate DNA unwinding. In studies with uniquely nicked duplex DNA, we present EM evidence that helicase II protein initiates DNA unwinding at the nick, with unwinding proceeding bidirectionally. We also show that helicase II protein initiates DNA unwinding at the blunt ends of linear DNA, rather than in internal regions. These data provide direct evidence that helicase II protein can initiate unwinding of duplex DNA at a nick, in the absence of auxiliary proteins. We propose that helicase II may initiate unwinding from a nick in a number of DNA repair processes.

Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA.

We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules. We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail. This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA. Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C). Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs. At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S. W. Matson [1986) J. Biol. Chem. 261, 10169-10175). This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo. The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined. Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E. coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS)

Large-scale purification and characterization of the Escherichia coli rep gene product.

We report a procedure for the large-scale purification of the Escherichia coli Rep protein, a helicase that is involved in the replication of the E. coli chromosome as well as a number of single-stranded bacteriophages. The procedure starts with E. coli cells harboring an overproducing plasmid, pRepO, in which the E. coli rep gene is under transcriptional control of the inducible lambda PL promoter (Colasanti, J., and Denhardt, D. T. (1987) Mol. Gen. Genet. 209, 382-390). The purification procedure results in greater than 98% pure Rep protein, which is free of contaminating nuclease activity, with yields of 40-50 mg of Rep protein/50 g of induced MZ-1/pRepO cells. We also show that cell death occurs upon inducing such a large overproduction of the E. coli Rep protein in MZ-1/pRepO. The Rep protein purified by this procedure has high specific single-stranded DNA-dependent ATPase activity, as well as helicase activity, with an apparent 3' to 5' directionality. The extinction coefficient of purified E. coli Rep protein is epsilon 280 = 1.16 +/- 0.04 ml mg-1 cm-1 (8.47 +/- 0.28 X 10(4) M-1 cm-1) in 10 mM Tris (pH 7.5), 20% (v/v) glycerol, 0.10 M NaCl at 25 degrees C. The solubility properties of the purified Rep protein have been examined as a function of glycerol, NaCl, MgCl2, ATP, and ADP concentrations at 25 and 37 degrees C (pH 7.5). Rep protein solubility decreases significantly with decreasing concentrations of glycerol and monovalent salt and increasing temperature; however, the presence of 1.5 mM ATP or ADP or MgCl2 at low NaCl concentrations increases the solubility. At 4 degrees C, in the presence of 20% glycerol and greater than or equal to 50 mM NaCl, the free Rep protein exists as a stable monomer under all conditions examined (+/- ATP and +/- MgCl2). The single-stranded DNA-dependent ATPase activity decreases with increasing glycerol concentration, such that in 25% (v/v) glycerol it has approximately 40% of its activity as compared to solutions that contain no glycerol. The dependence of the single-stranded DNA-dependent ATPase activity on salt concentration for a series of monovalent salts indicates the presence of both cation and anion effects, with decreasing activity in the order glutamate greater than acetate greater than chloride. The ability to obtain highly purified E. coli Rep protein in large quantities with relative ease will greatly facilitate physical characterizations of the protein and its interactions with DNA.