Cell Biology Exam 4: Chapter 13

DNA Replication and Repair

DNA duplicates by a process called ____ _________

DNA replication

the DNA replication machinery is also used for ___ _______

DNA repair

sense strand (what is it also known as & what does it contain)

• also known as coding, plus, or no-template strand

• contains codons & is same as mRNA except the T in DNA is replaced by U in the RNA

antisense strand (what is it also known as, what does it serve as, what is it complementary to, & what does it contain)

• also known as non-coding, minus, or template strand

• serves as a template for mRNA synthesis

• hence complementary to both the sense strands and mRNA (with U in RNA in place of T)

• the antisense/non-coding strand contains anti-codons

what are the two strands of the double helix held together by

• hydrogen bonds between the bases

• A & T (2 H bonds)

• C & G (3 H bonds)

Watson & Crick: replication

envisioned that replication occurred by gradual separation of the strands of the double helix

how does DNA replication take place & what is this called

DNA replication takes place by separation of the strands of the double helix, and synthesis of two daughter strands complementary to the two parental templates → semiconservative replication

what are the 3 proposed models of DNA replication

• conservative model

• semiconservative model

• dispersive model

conservative model

parental strands re-associate after acting as templates for new strands

semiconservative model

the 2 parental strands separate; each acts as template for new, complementary strand

dispersive model

each daughter strand contains a mixture of parental and new DNA

what were the experiments performed to determine the currently accepted mode of DNA replication & prokaryote or eukaryote

• Meselson and Stahl experiment (prokaryote)

• Herbert Tylor’s experiment (eukaryote)

Meselson & Stahl experiment (what did they culture, what were the nitrogens, steps of experiment, what was concluded)

• cultured E.Coli bacteria for several generations

• ¹⁵N was the heavy isotope of nitrogen and ¹⁴N was the lighter more common isotope of nitrogen

Steps

1. bacteria cultured in medium containing ¹⁵N

2. bacteria transferred to medium containing ¹⁴N

3. DNA sample centrifuged after 20 mins (1st replication → 1 band)

4. DNA sample centrifuged after 40 min (2nd replication → 2 bands)

• conclusion: DNA replication follows the semiconservative model 1 band for ¹⁴N (higher) and another band with ¹⁴N &¹⁵N (lower)

why is DNA replication called semiconservative

half of the parent structure is retained in each of the daughter duplexes

Herbert Tylor’s experiment (what was cultured, what compound was used, what were the findings)

• cultured mammalian cells were allowed to undergo replication in bromodeoxyuridine (BrdU) a compound that is incorporated into DNA in place of thymidine

• after 1 round of replication in BrdU → both chromatids of each chromosome conatined BrdU

• after 2 rounds of replication in BrdU → one chromatid of each chromosome was composed of 2 BrdU-containing strands, & the other chromatif was a hybrid consisting of a BrdU-containing strand and a thymidine-containing strand

replication in bacteria: where does it start & what occurs

starts at the origin site (origin of replication) where a number of proteins bind to initiate replication

replication in bacteria: replication forks (what they are & what direction replication proceeds)

• replication forks are points where a pair of replicating segments come together and join the nonreplicated segments

• replication proceeds bidirectionally

what occurs as DNA begins the unwinding process

tension is built up as DNA begins the unwinding process, and the DNA becomes positively supercoiled

DNA gyrase (what is it also called, what does it do, & what does it use)

• topoisomerase II

• relives tension by changing DNA into negatively supercoiled (underwound) DNA

• uses ATP hydrolysis

torsional stress caused when unwinding DNA strands: what happens & what relieves tension

• unseparated portion becomes more tightly wound

• topoisomerase or gyrase breaks & rejoins coiled strands ahead of replication and thereby relives tension

DNA polymerase

responsible for synthesizing new DNA strands from a DNA template

what does DNA polymerase require

a primer

what do primers provide

provides the 3’-OH terminus on which to add new nucleotides

what direction does DNA replication/polymerization occur

5’ to 3’ direction

can any of the 3 DNA polymerases in bacteria initiate DNA chains?

no

how does DNA polymerization occur in terms of the OH group and the phosphate group

the -OH group at the 3’ end of the primer carries out a nucleophilic attack on the 5’-phosphate of the incoming nuceloside triphosphate

where can DNA replication only add new strands

the phosphate group always gets added to the 3’-OH end

what is at the 5’ carbon and 3’ carbon of DNA

• 5’ → phosphate group

• 3’ → hydroxyl group

semidiscontinuous replication: how are both daughter strands synthesized

simultaneously

semidiscontinuous replication: the leading strand (direction & how it is synthesized)

• in the direction of the replication fork movement

• synthesized continuously

semidiscontinuous replication: the lagging strand (direction & how it is synthesized)

• in the opposite direction of the replication fork

• synthesized discontinuously

okazaki fragments (what they are & what are they joined by)

the small discontinuous fragments that the lagging strand is composed of which are joined by DNA ligase

DNA ligase

• seals fragments/strands

• seals Okazaki fragments into continuous strand

how is synthesis of each Okazaki fragment of the lagging strand intitated

short RNA fragments are used as removable primers in initiating synthesis of each Okazaki fragment of the lagging strand (primers introduce 3’-OH end)

why is synthesis of the lagging strand discontinous

• during replication the phosphate group can only add at the 3’-OH end and the lagging strand has a 5’ end to start

• that’s why the primers with 3’-OH ends need to be added

primase

an RNA polymerase that assembles short RNA primers

how are the RNA primers removed

by exonuclease activity of DNA polymerase I, which also fills in the gaps

what 2 things unwind the parental duplex and separate the two strands

• Helicase

• single-stranded DNA-binding (SSB) proteins

DNA polymerase III (what it is & what it does)

• primary replication enzyme

• synthesize successive fragments of the lagging strand

DNA polymerase II

DNA repair enzyme with a 3’ to 5’ exonuclease activity

what is DNA polymerase I involved in

DNA repair & also removes primers and replaces them with DNA

exonucleases

degrade nucleic acids by removing 5’ or 3’ terminal nucleotides

what are the exonuclease activities of DNA polymerase I

• 5’ → 3’: removes from 5’ end plays a role in removing the RNA primer

• 3’ → 5’: removes from the 3’ end removes mispaired nucleotides & maintains the accuracy of DNA synthesis

3’ → 5’ exonuclease of DNA polymerase I (what occurs during proofreading, what accounts for low error rates, how quick is replication)

• during proofreading, mismatched bases are excised

• careful selection of the nucleotide, proofreading, and mismatch repair account for low error rates in replication

• replication is rapid

exonuclease activity DNA polymerase I vs II

DNA polymerase I

• 5’ → 3’ (removing RNA primers)

• 3’ → 5’ (proofreading)

DNA polymerase II

• 3’ → 5’ (proofreading & backup repair)

beginning of replication prokaryotes vs eukaryotes

prokaryotes

• singe origin of replication

eukaryotes

• multiple origins of replication (the genome is very big so one origin of replication would be too slow)

how do eukaryotes replicate their genome

in small portions (replicons)

there are _____ DNA polymerases in eukaryotes

several

how do eukaryotic DNA polymerases elongate (what direction, what is required, & what activity do some DNA polymerases have)

• elongate in the 5’ to 3’ direction

• require a primer

• some have 3’ to 5’ exonuclease activity

functions: gyrase, primase, DNA ligase

• gyrase (topoisomerase I/II): relieves positive supercoils ahead of replication fork

• primase: synthesizes RNA primers

• DNA ligase: seals Okazaki fragments into continuous strand

where is the replication machinery stationary

in the nuclear matrix

replication foci (what they are & what do they demonstrate)

• replication foci are the sites in which replication forks are located

• demonstrate that replication activities do not occur randomly throughout the nucleus but are confined to distinct sites

Helicase

unwinds DNA

single-stranded DNA binding proteins

keep strands apart

what is DNA repair essential for

cell survival

what can create spontaneous alteration (lesions) in DNA

• ionizing radiation

• common chemicals

• UV radiation

• thermal energy from normal metabolism

fidelity

how accurate DNA copying is

3 distinct categories that the fidelity of DNA can be traced to

• accurate selection of nucleotides (base pairing rules)

• immediate proofreading

• post-replicative mismatch repair

what is formed within a DNA duplex following UV radiation

a pyrimidine dimer → tanning beds & prolonged sun exposure

what are the 4 DNA repair mechanisms

• nucleotide excision repair

• base excision repair

• mismatch repair

• double stranded break repair

base excision repair: what it does & what enzyme

• removes altered nucleotides that produce distortions of the double helix

• DNA glycosylases recognize the alterations and cleave the base from the sugar; they are specific for a particular type of altered base

• once removed, an endonuclease cleaves the DNA backbone & a polymerase fills the gap by inserting a nucleotide complementary to the undamaged strand

• DNA ligase seals the strand

DNA glycosylases: what they do & example

• DNA glycosylases recognize the alterations and cleave the base from the sugar; they are specific for a particular type of altered base

• ex.) hOGG1: DNA glycosylase; detects the oxidized form of guanine and it is able to fit into the active site of the enzyme

mismatch repair

the correction of mistakes that escape

what causes double-strand breaks

ionizing radiation (X-rays, gamma rays) along with some chemicals

by what pathways can double stranded breaks be repaired

• non-homologous end joining

• homologous recombination

double stranded breaks repair: nonhomologous end joining (NHEJ)

• ku proteins bind to the free, broken ends and catalyze a reaction to rejoin the broken ends

• mediated through DNA-dependent protein kinases

double stranded breaks repair: homologous recombination

• requires a homologous chromosome to serve as a template for repair of the broken strand

• more accurate than NHEJ

ATM protein kinases (what is it activated by, what does it do, & what does it activate)

• activated by double-strand breaks

• stops cell cycle

• activates DNA repair proteins

ATR protein kinases (what is it activated by, what does it do, & what does it prevent)

• activated by replication stress/single-stranded DNA

• stabilizes replication fork

• prevents collapse

what are skin cells with optimal levels of repair enzymes subject to

lesions that fail to be excised and repaired

skin cancer

disease promoted by deficiency or overworked DNA repair systems

colon cancer

due to mutations in mismatch repair genes