BIOL 311: Chapter 12

Steps for completing the lagging strand

1. Removal of RNA primer (DNA polymerase I) 2. Synthesis of DNA (DNA polymerase I) 3. Linking Okazaki fragments togther (DNA ligase)

5'->3' and 3'->5' exonuclease activity

5'->3': Cuts back DNA starting at 5' end, removes all oligos 3'->5': Cleaves mononucleotides from 3' end of DNA (proofreading). Most errors are corrected by this.

Mismatch repair

A system for recognizing the repairing erroneous insertion, deletion, and mis-incorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage.

What happens to histones during replication?

After DNA replication, the new reassembled octamers are a random mixture of old and new histones. isolate octomers, spin, and there's a single band; old octomers with heavy amino acids change the medium, induce replication and isolate the octomers, spin it and you get a broad band; octomers with mixture of old and new histones (heavy and light amino acids).

Okazaki fragments are associated with synthesis of: a. the leading strand. b. the lagging strand. c. both the leading and the lagging strands. d. single-stranded circular DNA.

B. The lagging strand

Rolling Circle Replication

Circular DNA Template Breakage of Nucleotide Strand 1 Replicon Unidirectional Products: One circular molecule and one linear molecule that may circularize

Theta Replication

Circular DNA Template No Breakage of Nucleotide Strand 1 Replicon Unidirectional or bidirectional Products: Two circular molecules

Elongation

DNA polymerase requires the 3'-OH to initiate synthesis. Can only add nucleotides to 3'-OH of preceding nucleotide Primase: an RNA polymerase that synthesizes a short RNA complementary to template strand. RNA polymerases do not require free 3'-OH for synthesis. Provides a terminus with a free 3'-OH Synthesis of primers: For initiation of leading strands and of each Okazaki fragment.

Licensing of DNA replication

Ensures that replication happens once and only once. 1. Binding of replication licensing factor to origin of replication. -MCM is a eukaryotic licensing factor that contains a DNA helicase. 2. Replication machinery initiate replication at licensed origins. -Licensing factor is removed after replication is initiated and cannot be re-licensed until after the cell completes mitosis. -Geminin binds and prevents MCM from binding again.

What do each of these proteins do? DNA polymerases DNA helicases DNA gyrases (topioisomerases) DNA primase Sing-stranded binding proteins

DNA polymerases: Add complementary nucleotides DNA helicases: Unwinds DNA double helix DNA gyrases (topioisomerases): Prevents over-coiling DNA primase: Makes RNA primer Sing-stranded binding proteins: Keeps DNA single-stranded

Direction of Synthesis

Determined by DNA polymerase activity. DNA polymerase adds to nucleotides to 3' OH of previous nucleotide Direction of synthesis 5' -> 3' Template is read 3' -> 5' Antiparallel strands get synthesized in different directions.

Meselson and Stahl Experiment

Grew E.coli in a medium containing heavy nitrogen (15N), and incorporated it into DNA ("heavy" DNA). Then shifted E. coli to medium containing 14N ("Lighter" DNA). Therefore, the newly synthesized DNA should be lighter. Separate the DNA using equilibrium density gradient centrifugation, and the DNA is separated according to density. Heavy DNA moves toward the bottom, and light DNA migrates to the top. They cultured bacteria in a 15N medium. 15N is a heavy isotope of nitrogen so the DNA synthesized is of heavy density. They then shifted the bacteria to a 14N medium, DNA was isolated at different times corresponding to replication cycles 0, 1, and 2. After one replication cycle, the DNA was all of intermediate density. This rules out the conservative replication model, which predicts that both heavy density DNA and light density DNA will be present, but none of intermediate density will be present. This result is consistent with the semiconservative replication model, which predicts that all DNA molecules will consist of one 15N-labeled DNA strand and one 14N-labeled DNA strand. The result does not rule out the dispersive replication model, which also predicts that all DNA will be of intermediate density, consisting of interspersed double-stranded 15N-labeled and 14N-labeled segments. After two replication cycles, two bands of DNA were seen, one of intermediate density and one of light density. This result is exactly what the semiconservative model predicts: half should be 15N-14N intermediate density DNA and half should be 14N-14N light density DNA. This result rules out the dispersive replication model, which predicts that after replication cycle 1, the DNA density of all DNA molecules will gradually become lower, so no intermediate density DNA should remain at replication cycle 2. The semiconservative model is correct.

DNA Polymerases I and III

I: 5' -> 3' polymerization, 3'->5' exonuclease (proofreading), and 5'->3' exonuclease. Removes and replaces primers. Not the major replication enzyme. III: 5' -> 3' polymerization, 3'->5' exonuclease (proofreading), and NO 5'->3' exonuclease. Elongates the DNA.

Leading and lagging strands

Leading: Continuous DNA synthesis 5' to 3' with the 5' end of the strand being synthesized coming off the replication fork Lagging: Discontinuous DNA synthesis 5' to 3' with the 3' end of the strand being synthesized coming off the replication fork Okazaki fragments: The fragments of the lagging strand that are later linked together with DNA ligase to make one continuous strand.

Linear Eukaryotic Replication

Linear DNA Template No Breakage of Nucleotide Strand Many Replicons Bidirectional Products: Two linear molecules

Initiation of DNA replication

OriC= E.coli origin Initiator protein (DnaA) binds to OriC and twists DNA and unwinds a small section. Helicase unwinds the DNA and SSBs bind.

What happens to nucleosomes during replication?

Nucleosomes disassemble at the replication fork, and reassemble after the fork passes. It's distributed to both helices, and new nucleosomes are also formed.

DNA Topioisomerases

Remove supercoils ahead of unwinding DNA Topo I: Makes single strand breaks which provides an axis of rotation. DNA on opposite side can spin independently. Topo II (DNA gyrase): Makes double strand breaks to relieve torsional strain that builds up ahead of replication fork. Adds negative supercoils of removes positive supercoils.

Telomers

Repetitive sequences at the ends of chromosomes. Prevent nucleases from degrading ends of linear DNA Prevents fusion of ends with other DNA Facilitates replication of the ends of linear DNA without loss of material. Contain short nucleotide sequence present as tandem repeats. Have sequence of 5'-(A or T)m Gn-3' m=1-4 n=2 or more

3 Proposed Models for DNA Replication

Semiconservative: The two strands of the parental molecule separate and each functions as a template for synthesis of a new, complementary strand. Conservative: The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. Dispersive: Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA.

Telomerase activity

Teloerase extends the DNA, filling in the gap due to the removal of the RNA primer.

Requirements for Replication

Template: Single stranded Primer Substrates (dNTPs) Enzymes and other proteins

1. Meselson and Stahl grew bacteria for many generations in medium containing heavy 15N, then shifted the bacteria into medium containing light 14N for one, two, or three rounds of DNA replication. The DNA was extracted after each round and the density of the DNA molecules was determined by density gradient centrifugation. After the first round of replication, Meselson and Stahl saw only one DNA band of density intermediate to DNA containing only 15N or 14N. After this observation, which hypothesis for DNA replication could be eliminated? a. conservative b. semiconservative c. dispersive d. None of the above could be ruled out

a. CONSERVATIVE

DNA polymerases in eukaryotic cells: alpha delta epsilon gamma

alpha primase, primes both leading and lagging strands function: Initiation of nuclear DNA synthesis and DNA repair; has primase activity delta Elongates the lagging strand function: lagging strand synthesis of nuclear DNA, DNA repair, and translesion DNA synthesis epsilon elongates the leading strand function: leading strand synthesis gamma function: Replication and repair of mitochondrial DNA 5' -> 3' polymerase activity: all four 3'->5' exonuclease activity: All but alpha

DNA replication in eukaryotes differs from replication in prokaryotes in that: a. DNA replication in eukaryotes is conservative, whereas in prokaryotes it is semiconservative. b. eukaryotes have bidirectional replication from an origin, whereas in prokaryotes replication proceeds in one direction from an origin. c.eukaryotic chromosomes have many separate origins of replication, whereas prokaryotic chromosomes have a single origin of replication. d. linear eukaryotic chromosomes are replicated by a mechanism called theta replication, whereas circular prokaryotic chromosomes are replicated by the rolling circle mechanism.

c. eukaryotic chromosomes have many separate origins of replication, whereas prokaryotic chromosomes have a single origin of replication.

During initiation of DNA replication in E. coli, what is the role of helicase? a. It binds to the origin and causes a short section of the double helix to unwind. b. It binds to and stabilizes the single-stranded DNA. c. It reduces torsional strain by making double-stranded DNA breaks ahead of the replication fork. d. It breaks hydrogen bonds between the two paired DNA strands.

d. It breaks hydrogen bonds between the two paired DNA strands.