RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30 microm s(-1)), with as many as 42,300 base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

The DNA in sperm and certain viruses is condensed by arginine-rich proteins into toroidal subunits, a form of packaging that inactivates their entire genome. Individual DNA molecules were manipulated with an optical trap to examine the kinetics of torus formation induced by the binding of protamine and a subset of its DNA binding domain, Arg6. Condensation and decondensation experiments with lambda-phage DNA show that toroid formation and stability are influenced by the number of arginine-rich anchoring domains in protamine. The results explain why protamines contain so much arginine and suggest that these proteins must be actively removed from sperm chromatin after fertilization.

No abstract

Microfluidic flow cells are used in single-molecule experiments, enabling measurements to be made with high spatial and temporal resolution. We discuss the fundamental processes affecting flow cell operation and describe the flow cells in use at present for studying the interaction of optically trapped or mechanically isolated, single DNA molecules with proteins. To assist the experimentalist in flow cell selection, we review the construction techniques and materials used to fabricate both single- and multiple-channel flow cells and the advantages of each design for different experiments.

Two transition proteins, TP1 and TP2, participate in the repackaging of the spermatid genome early in mammalian spermiogenesis, coincident with the first detectable changes in chromatin condensation. Using an optical trap and a two-channel flow cell to move single DNA molecules into buffer containing protein, we have measured the rates of DNA condensation and decondensation induced by the binding of Syrian hamster transition proteins TP1 and TP2 and protamines P1 and P2. The results show that both transition proteins condense free DNA, with rates similar to those of protamine 1 and 2. DNA molecules condensed with TP1 were significantly less stable than DNA condensed by protamine or by TP2. Experiments conducted with a peptide corresponding to the C-terminal 25 residues of TP2 showed that this domain is responsible for condensing DNA. Experiments conducted with two fragments of TP1 containing arginine and lysine residues demonstrated that DNA binding by TP1 must involve more than these basic sequences. Zinc facilitated the condensation of DNA by P2 but not by TP2. The dissociation rates of TP2 and P2 from DNA were not affected by the addition of zinc.The structure of chromatin is changed dramatically during the final stages of spermiogenesis in mammals as the spermatid's genome is condensed and inactivated by the sequential binding of several basic nuclear proteins (1). Although the most dramatic change in condensation occurs in late-step spermatids (2) when the protamines displace transition proteins TP1 and TP2 (3) and coil the DNA into toroidal subunits (4), the replacement of histones by these two transition proteins several days earlier coincides with the first appearance of a detectable change in the condensation state of chromatin (5, 6). Studies in the rat have demonstrated that the deposition of the two transition proteins in spermatid chromatin occurs sequentially, with TP2 appearing first in step 10 spermatids. TP1 was observed to appear ϳ24 h later in step 12 spermatids (7).Previous experiments have shown that TP2 binds preferentially to CG sequences, and the observation that TP2 appears early in spermatid chromatin (8) suggested that the function of TP2 may be to shut down transcription by binding to the CG islands that are associated with gene promoter domains. TP1, which appears a day later, has been reported to stimulate DNA repair of single-stranded breaks (9) and is suggested to function by binding to the breaks induced during the removal of the histones until they can be repaired (9, 10). Following the removal of the histones and the repair or ligation of the singlestranded breaks, protamines are synthesized and deposited on chromatin to complete the compaction of the chromatin and ensure the sperm genome remains inactive until it can be deposited inside an egg and reactivated.Protamines, on the other hand, are highly charged, argininerich proteins that bind to DNA in a nonspecific manner. Previous in vivo and in vitro studies have been carried out to determine how DNA is condensed by protamine...

Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

Protamine molecules bind to and condense DNA in the sperm of most vertebrates, packaging the sperm genome in an inactive state until it can be reactivated following fertilization. By using methods that enable the analysis of protamine binding to individual DNA molecules, we have monitored the kinetics of DNA condensation and decondensation by protamine 1 (P1) and synthetic peptides corresponding to specific segments of the bull P1 DNA binding domain. Our results show that the number of clustered arginine residues present in the DNA binding domain is the most important factor affecting the condensation and stability of the DNA-protamine complex prior to the formation of inter-protamine disulfide cross-links. The high affinity of P1 for DNA is achieved by the coordinated binding of three anchoring domains, which together in bull P1 contain 19 Arg residues. The single DNA molecule experiments show that sequences containing two or more anchoring domains have an off-rate that is at least 3 orders of magnitude slower than those containing a single domain. The use of Arg, rather than Lys residues, and the inclusion of Tyr or Phe residues in the hinge regions between anchoring domains provide additional stability to the complex.

No abstract