Chapter 34: DNA Replication

5. Match each term with its description. (a) DNApolymerase (b) Template (c) Primer (d) Helicase (e) Topoisomerase (f) Exonuclease (g) Primase (h) Okazaki fragment (i) DNA ligase (j) Telomerase 1. Unwinds the double helix 2. Crucial for proofreading 3. Uses ATP to join DNA fragments 4. Determines the sequence of a complementary nucleic acid 5. Faithfully replicates the DNA 6. Synthesizes a segment of RNA 7. Found on the lagging strand 8. The initial segment of a polymer that will be extended 9. Prevents the disappearance of the lagging strand 10. Relaxes or introduces supercoils

(a) 5; (b) 4; (c) 8; (d) 1; (e) 10; (f) 2; (g) 6; (h) 7; (i) 3; (j) 9

lagging strand

A newly synthesized strand of DNA at the replication fork that is initially synthesized as Okazaki fragments. See also leading strand.

leading strand

A newly synthesized strand of DNA at the replication fork that is synthesized continuously. See also lagging strand.

origin of replication

A particular sequence in a genome at which replication begins.

processivity

A property of an enzyme that enables it to catalyze multiple rounds of elongation or the digestion of a polymer while the polymer stays bound to the enzyme.

telomerase

A reverse transcriptase that contains its own template; a highly processive enzyme that elongates the 3′-ending strand of a telomere.

primase

A specialized RNA polymerase that synthesizes the RNA primers for DNA synthesis.

1. Define template and primer as they relate to DNA synthesis.

A template is the sequence of DNA or RNA that directs the synthesis of a complementary sequence. A primer is the initial segment of a polymer that is to be extended on which elongation depends.

DNA ligase

An enzyme that catalyzes the formation of a phosphodiester linkage between the 3′-hydroxyl group at the end of one DNA strand and the 5′-phosphoryl group at the end of another strand; it takes part in the synthesis, repair, and splicing of DNA.

topoisomerase

An enzyme that catalyzes the interconversion of topoisomers of DNA; can relax supercoiled DNA.

exonuclease

An enzyme that digests nucleic acids from the ends of the molecules, rather than at an internal site; exonucleases can be specific for digestion from the 3′ or 5′ ends of the nucleic acid.

2. Explain why DNA synthesis depends on RNA synthesis.

DNA polymerase cannot initiate primer synthesis. Consequently, an RNA polymerase, called primase, synthesizes a short sequence of RNA that is used as a primer by the DNA polymerase.

34.1 DNA Is Replicated by Polymerases

DNA polymerases are template-directed enzymes that catalyze the formation of phosphodiester linkages by the 3′-hydroxyl group's nucleophilic attack on the innermost phosphorus atom of a deoxyribonucleoside 5′-triphosphate. They cannot start strands de novo; a primer with a free 3′-hydroxyl group is required. Many DNA polymerases proofread the nascent product: their 3′ S 5′ exonuclease activity potentially edits the outcome of each polymerization step. A mispaired nucleotide is excised before the next step proceeds. In E. coli, DNA polymerase I repairs DNA and participates in replication. Fidelity is further enhanced by an induced fit that results in a catalytically active conformation only when the complex of enzyme, DNA, and correct dNTP is formed. Helicases prepare the way for DNA replication by using ATP hydrolysis to separate the strands of the double helix. Single-strand-binding proteins bind to the separated strands of DNA to prevent them from reannealing. Separation of the DNA into single strands introduces torsional stress in the DNA ahead of the separation. Topoisomerase relieves the stress by introducing supercoils.

34.2 DNA Replication Is Highly Coordinated

DNA replication in E. coli starts at a unique origin (oriC) and proceeds sequen- tially in opposite directions. More than 20 proteins are required for replication. An ATP-driven helicase unwinds the oriC region to create a replication fork. At this fork, both strands of parental DNA serve as templates for the synthesis of new DNA. A short stretch of RNA formed by primase, an RNA polymerase, primes DNA synthesis. One strand of DNA (the leading strand) is synthesized continuously, whereas the other strand (the lagging strand) is synthesized dis- continuously, in the form of 1-kb Okazaki fragments. Both new strands are formed simultaneously by the concerted actions of the highly processive DNA polymerase III holoenzyme, an asymmetric dimer. The discontinuous assem- bly of the lagging strand enables 5′ S 3′ polymerization at the molecular level to give rise to overall growth of this strand in the 3′ S 5′ direction. The RNA primer stretch is hydrolyzed by the 5′ S 3′ nuclease activity of DNA poly- merase I, which also fills gaps. Finally, nascent DNA fragments are joined by DNA ligase in a reaction driven by ATP. DNA synthesis in eukaryotes is more complex than in bacteria. Eukaryotes require thousands of origins of replication to complete replication in a timely fashion. A special RNA-dependent DNA polymerase called telomerase is responsible for the replication of the ends of linear chromosomes.

telomere

Each end of a chromosome; the DNA at the telomere consists of hundreds of repeats of a hexanucleotide sequence characteristic of the organism.

DNA polymerase

Enzymes that catalyze the template-directed, primer-dependent addition of deoxynucleotide units, using deoxynucleoside triphosphates as substrates, to the 3′ end of a DNA chain; chain growth is in the 5′-to 3′-direction; such enzymes replicate and repair DNA.

9. Why would replication come to a halt in the absence of topoisomerase II?

Eventually, the DNA would become so tightly wound that movement of the replication complex would be energetically impossible.

template

In DNA or RNA, a sequence that directs the production of a complementary sequence.

trombone model

In DNA replication, the lagging strand loops out to form a structure that acts somewhat as a trombone slide does, growing as the replication fork moves forward. When the polymerase on the lagging strand reaches a region that has been replicated, the sliding clamp is released and a new loop is formed.

primer

In the elongation of polymers, the initial segment of the polymer that is to be extended. In DNA synthesis, elongation depends on the primer.

3. What is an Okazaki fragment?

Okazaki fragments are short segments of DNA that are synthesized on the lagging stand of DNA. These fragments are subsequently joined by DNA ligase to form a continuous segment of DNA.

Okazaki fragment

Small fragments of DNA (approximately 1000 nucleotides) that are formed on the lagging strand at the replication fork of DNA synthesis and later joined; enable 5′ S 3′ polymerization at the nucleotide level while overall growth is in the 3′ S 5′ direction.

sliding clamp

The dimeric b2 subunit of DNA polymerase III through which the DNA template slides. The enzyme is able to move without falling off its substrate by clasping the DNA molecule.

replication fork

The site of DNA synthesis where the parental strands are separated and daughter strands complementary to each parent are synthesized.

4. Distinguish between the lead- ing and the lagging strands in DNA synthesis.

When DNA is being synthesized at the replication fork, the leading strand is synthesized continuously in the 5′-to-3′ direction as the template is read in the 3′-to-5′ direction. The lagging strand is synthesized as short Okazaki fragments.