The kinetics of spontaneous DNA branch migration.

Abstract

We discuss the role of proteins in promoting the branch migration step during homologous recombination.Genetic recombination involving the exchange of genetic information between two DNA molecules has been observed in virtually all organisms. An important intermediate in both homologous and site-specific recombination is the Holliday junction, the branch point connecting two duplex DNAs that are undergoing recombination. If the branch point is flanked by DNA sequence homology, the Holliday junction can spontaneously migrate in either direction by the exchange of hydrogen bonds between the bases in homologous DNA strands. This process is known as branch migration and is an important step in genetic recombination. In homologous recombination, the extent of branch migration affects the amount of genetic information that is transferred between two homologues. In site-specific recombination, such as in the integration of bacteriophage A into the Escherichia coli chromosome, branch migration in the region of homology shared between donor and recipient sites is a prerequisite for subsequent steps in the recombination pathway. Early estimates of the rate of branch migration yielded a step rate of several thousand bases per sec (1), a rate that was robust enough to be accommodated within the time scale of recombination in bacteriophages. This suggested that branch migration could occur spontaneously during recombination without the need for proteins to facilitate this step. Later, several groups studying cruciform transitions in supercoiled DNA obtained data suggesting that the rate of spontaneous branch migration might be significantly slower (2-4).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2021In addition to knowing the inherent rate of branch migration, it is also critical to know whether spontaneous branch migration can traverse sequence heterology such as mismatches, insertions, and deletions since homologous recombination usually involves the exchange of DNA strands between two similar but not identical duplexes. We recently observed that a single base mismatch was sufficient to slow the overall rate of branch migration (5). Moreover, this attenuation by sequence heterology was more pronounced in magnesium than in sodium, suggesting that branch migration is influenced by metal ions.To clarify questions concerning the intrinsic rate of branch migration, we have developed an improved assay for branch migration to determine kinetic parameters of spontaneous DNA branch migration as a function of temperature and ionic conditions. We observe that branch migration in magnesium is '1000 times slower than was previously reported. In the absence of magnesium, branch migration is 3 orders of magnitude faster. We discuss the relationship between the structure of the Holliday junction and rates of branch migration and the need for proteins to promote branc...

Effect of DNA Supercoiling on the Geometry of Holliday Junctions

“…8b). This finding is consistent with the model in which unfolding of Holliday junction is required for branch migration (19,27,30,31). …”

Branch migration through DNA sequence heterology

“…in the presence of free Mg 2 , is much too slow to account for heteroduplex DNA formation within biologically relevant time-scales. This is due to the random walk nature of spontaneous branch migration, in which the junction has an equal probability to move forwards or backwards at each step as well as an intrinsically slow step-rate of several steps per second at 37 C (Panyutin & Hsieh, 1994). Similar rates of spontaneous branch migration have been attained using a variety of approaches (Fujitani & Kobayashi, 1995;Kirby et al, 1997;Muller et al, 1992;Mulrooney et al, 1996).…”

“…Branch migration through a single base-pair heterology was essentially blocked in the presence of Mg 2 at 37 C ( Figure 2B). Under these conditions, the intrinsic rate of branch migration is quite slow, of the order of several steps per minute (Panyutin & Hsieh, 1994). In the absence of Mg 2 ( Figure 2C), branch migration through a single base-pair heterology at 37 C was still blocked, despite an almost thousandfold increase in the rate of branch migration.…”

“…is accelerated approximately tenfold for each 10 C increase in temperature (Panyutin & Hsieh, 1994). Thus, branch migration of a Holliday junction through 60 bp containing a single base-pair heterology within a time-frame of a couple of hours required a 10,000-fold increase in the intrinsic rate of branch migration.…”

“…Due to secondary structure in the phage DNA that affected both the annealing and branch migration steps, a more extensive study of branch migration through sequence heterology was not possible. Here, we examine the effect of sequence heterology on branch migration using substrates designed to study the kinetics of spontaneous branch migration (Figure 1: Panyutin et al, 1995;Panyutin & Hsieh, 1994). Each substrate contains a 61 bp homologous duplex region and two heterologous, single-strand tails each 20 nt long.…”