The two strands of the parental DNA helix must be unwound in order for the polymerase to read each template and synthesize the new complementary strand. Enzymes that catalyze this separation are called DNA helicases. They catalyze the unwinding of the DNA duplex as the replication fork moves, usually using the energy of ATP hydrolysis to drive the process. Two molecules of ATP are hydrolyzed for each base pair that is unwound. Their activity can be measured biochemically by the conversion of duplex DNA to single-stranded DNA.
DNA helicases also have a second activity. They can move along single stranded DNA with a specific polarity. The polarity of movement can be measured by an in vitroassay using a substrate in which two distinctive short, labeled strands are in duplex with the two ends of a longer strand (Figure 5.22). A helicase will bind initially to the single-stranded portion of the substrate DNA and track along it until it meets a duplex region, at which point it will catalyze the unwinding of the duplex. Only one or the other of the short, labeled strands will be displaced by the helicase, depending on the direction of tracking along the single-stranded region. The displacement of molecule A in the Figure 5.22A shows that this helicase (DnaB) moved in a 5' to 3' direction along the single stranded DNA to reach the duplex portion including A. Fragment A was then displaced by the helicase activity.
Figure 5.22: An assay for direction of helicase tracking along a single stranded region.
Figure 5.22B shows the results of the tracking assay for a helicase called PriA. In what direction does it track along the single-stranded DNA?
Seven helicases have been isolated from E. coli. A distinctive function has not been defined for all of them, nor is it completely clear which act at the replication fork. The principal helicases for E. colichromosomal replication appear to be PriA and DnaB, which are also used in the machinery for making primers. An additional helicase that will be considered in Chapter 7 is helicase II, or UvrD, used in repair of DNA.
Once the two strands of the parental DNA molecule have been separated, they must be prevented from reannealing. Coating the single-stranded DNA with single-stranded DNA binding protein (SSB) does this (Figure 5.21).SSB from E. coli is a homotetramer, which a complex of four identical monomeric subunits. The monomeric protein subunits are 74 kDa; they are encoded by the ssbgene. Mutants in ssbstop DNA synthesis immediately, and hence they are needed for elongation. Such loss-of-function mutants are also defective in repair and recombination of DNA. SSB binds cooperatively to single‑stranded DNA, and in this form the single-stranded DNA cannot anneal to its complementary strand. An analogous protein found in eukaryotes is Replication Factor A, or RFA. This is a heterotrimer, i.e., composed of three different subunits.
The unwinding of the DNA strands by helicases affects the overall topology of the DNA (Figure 5.23). For instance, for every 10 base pairs that are unwound (DT=-1), there will be a compensatory increase in writhing (DW=+1) unless the linking number is changed. As discussed earlier in Chapter 2, enzymes that can change the linking number are topoisomerases. These enzymes act as swivels during replication to relieve the topological strain. In E. coli, this swivel is the enzyme gyrase (Table 5.3, Figure 5.23). This topoisomerase II uses the energy of ATP to make a double-strand break in a DNA molecule, passes a different portion of the DNA through the break, and reseals the break. The direction of passage is such that a negative superhelical turn is introduced, thereby counteracting the positive change in W that occurs when DNA is unwound. Compounds that inhibit gyrase, such as naladixic acid or the coumarins, also block DNA synthesis, showing the critical role for gyrase in this process. In mammalian cells, either topoisomerase I or topoisomerase II can be used as the swivel. Topoisomerase I makes a single stranded nick in supercoiled DNA, thereby allowing the supercoils to relax.
Figure 5.23. Changes in topology of the DNA during replication. The ATP-dependent untwisting (negative DT) catalyzed by DNA helicases causes a compensatory change in writhing (positive DW), which is relieved by the action of a topoisomerase II, such as DNA gyrase. Gyrase catalyzes an ATP-dependent, three-step reaction, cleaving the two strands of DNA, passing another part of the DNA duplex through the break and re-sealing the break. The action of gyrase generates a negative DW to balance the positive DW from the action of helicase. X and Y mark two different regions of the DNA molecule. A gray arrow indicates the direction of duplex movement through the break. Gyrase is a tetramer, and is shown as four pink balls.