O

S m xti x-ray film exposed to agarose get erty of many DNA-binding proteins, see Box 16-4, Concentration, Affinity, and Cooperative Binding.J Once covered with SSB, ssDNA is held in an elongated state that facilitides its use as a template for DNA or RNA primer synthesis.

SSB interacts with ssDNA in a sequence-independent mariner. SSBs primarily contact ssDNA through electrostatic interactions with

binding of additional SSBs O

FIGURE 8-14 Binding of single-stranded binding protein (SSB) to DNA. (a) A limiting amount of SSB ts bound to four of the nine ssDNA molecules shown, (b) As more SSB binds to DNA it preferentially binds adjacent to previously bound SSB molecules Only after SSB has completely coated the initially bound ssDNA molecules does binding occur on other molecules. Note that when ssDNA is coated with SSfi, it assumes a more extended conformation that inhibrts the formation of intramolecular base pairs binding of additional SSBs O

FIGURE 8-14 Binding of single-stranded binding protein (SSB) to DNA. (a) A limiting amount of SSB ts bound to four of the nine ssDNA molecules shown, (b) As more SSB binds to DNA it preferentially binds adjacent to previously bound SSB molecules Only after SSB has completely coated the initially bound ssDNA molecules does binding occur on other molecules. Note that when ssDNA is coated with SSfi, it assumes a more extended conformation that inhibrts the formation of intramolecular base pairs

restriction enzyme site

the phosphate backbone and stacking interactions with the DNA bases. In contrast to sequence specific DNA-binding proteins, SSBs make few, if any, hydrogen bonds to the ssDNA bases.

Topoisomerases Remove Supercoils Produced by DNA Unwinding at the Replication Fork

As the strands of DNA are separated at the replication fork, the double-stranded DNA in front of the fork becomes increasingly positively supercoiled (Figure 8-15). This accumulation of supercoils is the result of DNA helicase eliminating the base parts between the two strands. If the DNA strands remain unbroken, there can be no reduction in linking number (the number of times the two DNA strands are intertwined) to accommodate this unwinding of the DNA duplex (see Chapter 6). Thus, as the DNA helicase proceeds, the DNA must accommodate the same linking number within a smaller and smaller number of base pairs. Indeed, for the super-helicity to remain the same, one DNA link must be removed approx-

FI cure 8-15 Action of topoisomerase at the replication fork. As positive supercoils accumulate in front of the replication fork, topoisomerases rapidly remove them. In this diagram, the action of Topo II removes the positive supercoi! induced by a replication fort; Gy passtng one pert of the unrepltcated dsDNA through a double-stranded break in a nearby unrepealed region, the positive supercoils can be removed. It is worth noting Si at this change would reduce the linking number by two and thus would oniy have to occur once every 20 bp replicated. Although the action of a type 11 topoisomerase is illustrated here, type I topoisomerases can also remove the positive supercoils generated by the replication focfc.

replication machinery replication replication machinery replication break DNA

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