Lambda exonuclease processively degrades one strand of duplex DNA, moving 5′-to-3′ in an ATPindependent fashion. When examined at the single-molecule level, the speeds of digestion were nearly constant at 4 nanometers per second (12 nucleotides per second), interspersed with pauses of variable duration. Long pauses, occurring at stereotypical locations, were strand-specific and sequence-dependent. Pause duration and probability varied widely. The strongest pause, GGCGATTCT, was identified by gel electrophoresis. Correlating single-molecule dwell positions with sequence independently identified the motif GGCGA. This sequence is found in the left lambda cohesive end, where exonuclease inhibition may contribute to the reduced recombination efficiency at that end.Exonucleases are integral components of many genetic repair and recombination pathways (1). Processive 5′→3′ exonucleases generate DNA intermediates with long 3′-overhangs involved in these pathways (1, 2); in bacteriophage lambda, the Red α gene encodes the 24-kD subunit of such a nuclease (3). The structure of lambda exonuclease (λ-exo) consists of a toroidal homotrimer of subunits, with a tapered central channel sufficient to admit doublestranded DNA (dsDNA) at its entrance but only wide enough to pass single-stranded DNA (ssDNA) through its exit (4). A topological constraint may therefore underlie the high processivity of λ-exo (>3000 base pairs, where 1 bp corresponds to a helical rise of 0.338 nm along dsDNA) (5). λ-exo digestion requires Mg 2+ , but does not require ATP or GTP, and proceeds along DNA at rates of 2 to 3.5 nm/s, as determined by bulk biochemical studies (6, 7).Here, we developed a novel high-resolution, single-molecule assay to study λ-exo motion by optical trapping nanometry. Besides measuring individual translocation rates, single-molecule studies provide insights into other processes that are obscured by bulk averages. For example, certain processive DNA enzymes pause for variable intervals, a behavior that may reflect offpathway branches in the reaction cycle. Pausing behavior has been observed in single-molecule ‡To whom correspondence should be addressed.

Lambda exonuclease processively degrades one strand of double-stranded DNA (dsDNA) in the 5"-3" direction. To understand the mechanism through which this enzyme generates high processivity we are analyzing the first step in the reaction, namely the interaction of lambda exonuclease with the ends of substrate DNA. Endonuclease mapping of lambda exonuclease bound to DNA has shown that the enzyme protects approximately 13-14 bp on dsDNA, and no nucleo-tides on the single-stranded tail of the DNA product. We have developed a rapid fluorescence-based assay using 2-aminopurine and measured the steady-state rate constants for different end-structures of DNA. The relative k(cat)for 5" ends decreases in the order 5" recessed > blunt >> 5" overhang. However, k(cat)/K(m)remains relatively constant for these different structures suggesting they are all used equally efficiently as substrates. From these data we propose that a single-stranded 5" overhang end can bind non-productively to the enzyme and the non-hydrolyzed strand is required to aid in the proper alignment of the 5" end. We have also measured the length-dependence of the steady-state rate para-meters and find that they are consistent with a high degree of processivity.

We describe the purification to near homogeneity of a single-stranded DNA binding protein from 0-18-h embryos of Drosophila melanogaster. Drosophila SSB (D-SSB) is a heterotrimer with subunits of molecular weight of 70,000, 30,000, and 8000. It has a Stokes radius of 48.6 +/- 2 A and s20,w = 5.0 +/- 0.2 S. The interaction of D-SSB with ssDNA was examined by the quenching of intrinsic protein fluorescence. The binding site size was determined to be n = 22 +/- 4 nucleotides with a maximum quenching Qm = 35 +/- 3%. Equilibrium titrations indicate that D-SSB binds with low cooperativity, omega = 10-300, and high apparent affinity, K omega = (0.7-5) x 10(7) M-1, at 225 mM NaCl. Sedimentation of D-SSB bound to small oligonucleotides demonstrates that D-SSB does not require protein association for binding. D-SSB stimulates the extent and processivity of DNA synthesis of its cognate DNA polymerase alpha. On the basis of these properties, we conclude that D-SSB is the Drosophila cognate of the human and yeast SSB/RP-A proteins.

A number of terminal phosphate-labeled nucleotides with three or more phosphates and with varied length linkers attached between the terminal phosphate and the dye have been synthesized. These nucleotides have been tested as substrates for different DNA and RNA polymerases. We have also explored their utility in DNA sequencing, SNP analysis, nucleic acid amplification, quantitative PCR, and other biochemical assays.

DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster. This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication. The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells. This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA. By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase o-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A. In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase a-primase. These results collectively indicate that many of the specific functional interactions among T antigen, polymerase ei-primase, and RP-A are conserved from primates to Drosophila species. Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.DNA replication of the simian virus 40 (SV40) minichromosome is an important model system for the analysis of bidirectional double-stranded DNA replication in higher eukaryotes. Replication of the SV40 genome utilizes the host replication machinery in conjunction with a single virus-encoded protein, the large tumor antigen (T antigen). The development of a cell extract that is capable of replicating double-stranded DNA in the presence of an SV40 origin of replication has led to the identification of the components of the eukaryotic replication machinery and the characterization of their biochemical functions (see, for instance, references 9, 19, 37, 40, and 43 known as an Okazaki fragment. The resulting template-primer junction is recognized by a second multiprotein complex composed of replication factor C and proliferating cell nuclear antigen, both of which are accessory proteins for DNA polymerase 8. Binding of DNA polymerase 8 to this complex then leads to a polymerase switching step, in which polymerase 8 replaces polymerase at-primase as the leading-strand polymerase. Both polymerase ot-primase and polymerase 8 cooperate to synthesize Okazaki fragments on the lagging-strand template during the elongation phase of the replication reaction.SV40 DNA replication has been considered...