Enzymatic Activity Of

DNA helicases bind to single-stranded DNA (ssDNA) and translocate along the substrate either in a 5' to 3' or in a 3' to 5' direction using nucleoside triphosphate (NTP) hydrolysis for energy. Therefore, DNA helicases are defined by two biochemical activities: (a) DNA-dependent or DNA-stimulated ATPase activity and (b) NTP- or deoxyribonucleoside triphosphate (dNTP)-dependent strand-displacement activity, invariably with Mg2+ as a cofactor. BLM has been expressed in and purified from yeast and has been shown to exhibit both of these activities (65,71). In the strand-displacement assay, a standard substrate (Fig. 4) is prepared that contains a radiolabeled oligonucleotide annealed to M13 ssDNA or another oligonucleotide. BLM unwinds this type of substrate when a 3' single-stranded tail is available, indicating that BLM moves along ssDNA in a 3' to 5' direction, as do other RecQ helicases.

Compared to the standard substrate, purified recombinant BLM protein is more active in unwinding several unusual DNA substrates in vitro, including G4 DNA (72), triplex DNA (73), Holliday junctions (74), and D-loops (75). G4 DNA is a structure of four parallel DNA strands that are stabilized by Hoogsteen bonds between guanines. Triplex DNA can be generated when a third strand composed of either pyrimidines or purines lies in the major groove of duplex sequences. A Holliday junction is a four-way junction of two DNA duplexes that is formed during homologous recombination. A D-loop is a recombination intermediate that forms when the end of a ssDNA molecule invades and anneals with the homologous sequences of another DNA duplex. Other RecQ helicases exhibit similar capabilities, and the WRN helicase in particular can unwind G4 DNA (76), triplex DNAs (73), and Holliday junctions (77). (D-loops have yet to be tested.) The sub-

Figure 4 Selected substrates used for helicase assays. Conventional helicase substrates include the linear duplexes with 3' or 5' single-stranded overhangs and the circular substrates with or without nonhomologous tails. More unusual substrates that BLM binds and unwinds preferentially are the tetrahelical G4 DNA, the X junction, which resembles a Holliday junction, and D-loops, which resemble the joint molecules made by Rad51. Typically, one of the oligonucleotides is radiolabeled either with T4 polynucleotide kinase or with the Klenow fragment, and a annealing reaction is performed. Before incubating the substrate with enzyme, the substrate is purified. The products of the reaction are detected by polyacrylamide gel electrophoresis.

Figure 4 Selected substrates used for helicase assays. Conventional helicase substrates include the linear duplexes with 3' or 5' single-stranded overhangs and the circular substrates with or without nonhomologous tails. More unusual substrates that BLM binds and unwinds preferentially are the tetrahelical G4 DNA, the X junction, which resembles a Holliday junction, and D-loops, which resemble the joint molecules made by Rad51. Typically, one of the oligonucleotides is radiolabeled either with T4 polynucleotide kinase or with the Klenow fragment, and a annealing reaction is performed. Before incubating the substrate with enzyme, the substrate is purified. The products of the reaction are detected by polyacrylamide gel electrophoresis.

strate specificities of the BLM and WRN helicases presently cannot be used to explain the differences between BS and WS cells.

Of the 11 missense mutations found in BS-affected persons, all occur either in the helicase domain or in a 50-amino acid stretch of the RecQ C-terminal extended homology region. Experimentally, two mutations have been studied in some detail (65). One mutation changes the glutamine at residue 672 to an argi-nine (Q672R); this glutamine lies 10 amino acid residues N-terminal of helicase motif I, and it is conserved in many RecQ helicases. The other mutation replaces a conserved cystine at residue 1055 with a serine (C1055S). Two mutant BLM proteins each containing the Q672R and C1055S substitutions were expressed in and purified from yeast, and their biochemical activities were measured. The BLM Q672R protein had 40% of the normal DNA-dependent ATPase activity and lacked detectable DNA strand-displacement activity, and the BLM C1055S lacked both of these activities. By extension, we expect the other missense alleles in the helicase and C-terminal extended homology regions to either reduce or abolish the helicase activity of BLM.

Measurement of the SCE frequencies in BS cells in which different experimentally mutated BLM proteins have been stably expressed is the most powerful method of determining the function of a mutant BLM in vivo. Particularly useful for this purpose are the simian virus (SV40)-transformed BS fibroblasts, GM08505, which can proliferate in culture indefinitely, transfect easily, and exhibit the characteristic high-SCE phenotype. These cells harbor the blmAsh null allele at BLM, and by Western analysis BLM protein is undetectable. Stable expression of a normal BLM protein in GM08505 cells resulted in a correction of the high-SCE phenotype nearly to normal (78). In contrast, expression of either of the two missense mutant BLM proteins (Q672R and C1055S) failed to correct the high-SCE phenotype (65). Since the mutant proteins are defective helicases, their inability to rescue the high-SCE phenotype of BS cells indicated that the helicase activity is required for the cellular function of normal BLM.

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