Chapter 10 Flashcards

Question

All polymerases require preexisting DNA with two essential components. What are they? What do they do?

Answer 1

1. A primer function: The primer DNA provides a terminus with a free 3′-OH to which nucleotides are added during DNA synthesis. They catalyze the formation of a phosphodiester bridge between the 3′-OH at the end of the primer DNA chain and the 5′-phosphate of the incoming deoxyribonucleotide. 2. A template function: The template DNA provides the nucleotide sequence that specifies the complementary sequence of the growing DNA chain.

Answer 2

1. 5′ - 3′ polymerase activity 2. 3′ - 5′ exonuclease activities 3. 5′ - 3′ exonuclease **that is active only on single-stranded DNA**

Answer 3

1. 5 different types: DNA pol I: DNA repair enzymes DNA pol II: DNA repair enzymes and replication of damaged DNA DNA pol III: a complex enzyme composed of many different subunits DNA pol IV: replication of damaged DNA DNA pol V: replication of damaged DNA

Answer 4

Around 15: -DNA polymerases (α, δ, and/or ε) work together to carry out the semiconservative replication of nuclear DNA -DNA polymerase γ (gamma) is responsible for the replication of DNA in mitochondria

Answer 5

1. The catalytic core: -Contains three subunits: α (the dnaE gene product) ε (the dnaQ product) θ (the holE product) Addition of the τ subunit (the dnaX product) results in dimerization of the catalytic core and increased activity 2.The catalytic core synthesizes rather short DNA strands because of its tendency to fall off the DNA template. 3. The holoenzyme: (complete form) α, ε, θ, β, δ, γ The β-dimer forms a ring that encircles the replicating DNA molecule and allows DNA polymerase III to slide along the DNA while remaining tethered to it.

Answer 6

1. Scanning the termini of nascent DNA chains for errors and correcting them 2.This process is carried out by the 3′ - 5′ exonuclease activities of DNA polymerases

Answer 7

The 3′ - 5′ exonuclease activity of the DNA polymerase clips off the unpaired base or bases When an appropriately base-paired terminus is produced, the 5′ 3′ polymerase activity of the enzyme begins resynthesis by adding nucleotides to the 3′ end of the primer strand

Answer 8

In monomeric enzymes like DNA polymerase I of E. coli, the 3′ 5′ exonuclease activity is built in. In multimeric enzymes, the 3′ - 5′ proofreading exonuclease activity is often present on a separate subunit. In the case of DNA polymerase III of E. coli, this proofreading function is carried out by the ε subunit.

Answer 9

DNA polymerases γ, δ, and ε contain 3′ 5′ proofreading exonuclease activities Polymerases α and β lack this activity

Answer 10

1. It is carried out by the primosome 2. A protein complex containing DNA primase and DNA helicase 3a. The primosome moves along a DNA molecule, powered by ATP. b. As it proceeds, DNA helicase unwinds the parental double helix, and DNA primase synthesizes the RNA primers. c. The RNA primers are covalently extended with the addition of deoxyribonucleotides by DNA polymerase III. d. DNA topoisomerases provide transient breaks in the DNA that serve as swivels for DNA unwinding and keep the DNA untangled. e. Single-strand DNA binding protein coats the unwound DNA and keeps it in an extended state for DNA polymerase III. f. The RNA primers are replaced with DNA by DNA polymerase I. g. The single-strand nicks left by polymerase I are sealed by DNA ligase

Answer 11

1. The replisome 2. The replisome contains: -The DNA polymerase III holoenzyme -Two catalytic cores (one replicates the leading strand, the second the lagging strand) -The primosome 3. For the two catalytic cores of the polymerase III holoenzyme to synthesize the nascent leading and lagging strands, the lagging strand is thought to form a loop from the primosome to the second catalytic core of DNA polymerase III (refer to the photo of the e.coli replisome)

Answer 12

1. At variable sites within regions called terA and terB 2. They block the movement of replication forks advancing in the counterclockwise and clockwise directions, respectively. 3. DNA topoisomerases or special recombination enzymes then facilitate the separation of the nascent DNA molecules.

Answer 13

1. A mechanism for replicating circular DNA 2. It is used by: -Viruses to duplicate their genomes -Bacteria to transfer DNA from donor cells to recipient cells during genetic exchange -Amphibians to amplify extrachromosomal DNAs carrying clusters of ribosomal RNA genes during oogenesis 3. One parental circular DNA strand remains intact and rolls while serving as a template for the synthesis of a new complementary strand. 4. Replication is initiated when a sequence-specific endonuclease cleaves one strand at the origin, producing 3′-OH and 5′-phosphate termini. The 5′ terminus is displaced from the circle as the intact template strand turns about its axis. Covalent extension occurs at the 3′-OH of the cleaved strand. 5. It can produce either single-stranded or double-stranded progeny DNAs. 6. Single-stranded progeny molecules are produced by site-specific cleavage of the single-stranded tails at the origins of replication and recircularization of the resulting unit-length molecules. Double-stranded progeny molecules, the singlestranded tails are used as templates for the discontinuous synthesis of complementary strands prior to cleavage and circularization.

Answer 14

1. DNA replication is restricted to the S phase of the cell cycle (synthesis) 2. G1 phase (immediately following the completion of mitosis) S phase G2 phase (preparation for mitosis) M phase (mitosis) 3. Entry into S phase and entry into mitosis. These checkpoints help to ensure that the DNA replicates once and only once during each cell division

Answer 15

1. Polymerase α (Pol α) Polymerase δ (Pol δ) Polymerase ε (Pol ε) 2a. Pol α: -The initiation of replication at origins -The priming of Okazaki fragments during the discontinuous synthesis of the lagging strand. -Exists in a stable complex with DNA primase; (The primase synthesizes the RNA primers, which are extended with deoxyribo nt. by Pol α to produce an RNA–DNA chain) b. Pol δ: -Completes the replication of the lagging strand -Must interact with proteins PCNA and Rf-C to be active. c. Pol ε: -Catalyzes the replication of the leading strand 3. PCNA: (proliferating cell nuclear antigen) -A sliding clamp that tethers Pol δ to the DNA to allow processive replication (prevents DNA pol from falling off). PCNA is a trimeric protein that forms a closed ring. 4. Rf-C: (replication factor C) -Required for PCNA to load onto the DNA. -Induces a change in the conformation of PCNA that allows it to encircle DNA, providing the essential sliding clamp

Answer 16

1. Pol δ and ε do not have 5′ - 3′ exonuclease activity; thus, they cannot remove RNA primers like DNA polymerase I of E. coli. 2. RNA primers are excised by two nucleases: -Ribonuclease H1 (degrades RNA present in RNA–DNA duplexes) -Ribonuclease FEN-1 (F1 nuclease 1).

Answer 17

1. Nap-1 (nucleosome assembly protein-1) CAF-1 (chromatin assembly factor-1) 2a. Nap-1: -transports histones from their site of synthesis in the cytoplasm to the nucleus b. CAF-1: -Carries them to the chromosomal sites of nucleosome assembly -Delivers histones to the sites of DNA replication by binding to PCNA

Answer 18

1. An RNA-containing enzyme called telomerase which forestalls the shortening of chromosome ends by protecting them from degradation. 2.TTAGGG 3. It recognizes this G-rich telomere sequence on the 3′ overhang and extends it 5′ - 3′ one repeat unit at a time. -It does not fill in the gap opposite the 3′ end of the template strand; it extends the 3′ end of the template strand. -It contains a built-in RNA template.