Unwinding
Because DNA synthesis requires a single-stranded template, and therefore double-stranded DNA must be unwound before DNA synthesis can take place, the cell relies on several proteins and enzymes to accomplish the unwinding.
Figure 9.10: DNA helicase unwinds DNA by binding to the lagging-strand template at each replication fork and moving in the 5′→3′ direction.
DNA HELICASE A DNA helicase breaks the hydrogen bonds that exist between the bases of the two nucleotide strands of a DNA molecule. Helicase cannot initiate the unwinding of double-stranded DNA; the initiator proteins first separate DNA strands at the origin, providing a short stretch of single-stranded DNA to which a helicase binds. Helicase binds to the lagging-strand template at each replication fork and moves in the 5′→3′ direction along this strand, thus also moving the replication fork (Figure 9.10).
SINGLE-STRAND-BINDING PROTEINS After DNA has been unwound by helicase, single-strand-binding proteins (SSBs) attach tightly to the exposed single-stranded DNA (see Figure 9.10). These proteins protect the single-stranded nucleotide chains and prevent the formation of secondary structures that interfere with replication. Unlike many DNA-binding proteins, SSBs are indifferent to base sequence: they will bind to any single-stranded DNA. Single-strand-binding proteins form tetramers (groups of four); each tetramer covers from 35 to 65 nucleotides.
DNA GYRASE Another protein essential for the unwinding process is the enzyme DNA gyrase, a topoisomerase. As discussed in Chapter 8 and the introduction to this chapter, topoisomerases control the supercoiling of DNA. They come in two major types: type I topoisomerases alter supercoiling by making single-strand breaks in DNA, while type II topoisomerases create double-strand breaks. DNA gyrase is a type II topoisomerase. In replication, DNA gyrase reduces the torsional strain (torque) that builds up ahead of the replication fork as a result of unwinding (see Figure 9.10). It reduces torque by making a double-strand break in one segment of the DNA helix, passing another segment of the helix through the break, and then resealing the broken ends of the DNA. This action removes a twist in the DNA and reduces the supercoiling.
CONCEPTS
Replication is initiated at an origin of replication, where initiator proteins bind and cause a short stretch of DNA to unwind. DNA helicase breaks hydrogen bonds at a replication fork, and single-strand-binding proteins stabilize the separated strands. DNA gyrase reduces the torsional strain that develops as the two strands of double-helical DNA unwind.
CONCEPT CHECK 3
Place the following components in the order in which they are first used in the course of replication: helicase, single-strand-binding protein, DNA gyrase, initiator proteins.
Initiator proteins, helicase, single-strand-binding protein, DNA gyrase