Ch. 10 - Biosynthesis of Nucleic Acids-Replication

challenges in DNA replication

1. continuous unwinding & separation of the 2 DNA strands

2. must protect unwound portions of DNA from being attacked by nucleases

3. synthesis of DNA from 5’ to 3’

4. guard against errors—make sure right bases are added

how long does bacteria take to replicate?

20 mins

3 ways DNA can be replicated

1. semiconservative

2. conservative

3. dispersive model

semiconservative model

each daughter strand has 1 template strand & 1 newly synthesized one

• hybrids make 1 fully new & 1 that’s half-half

conservative model

1 daughter fully parental, 1 daughter fully newly synthesized

• only helices that contain fully parental strands make both

dispersive model

each daughter helix has some parental, some newly synthesized

• gradually less & less parental pieces of DNA

experiment for semiconservative replication

1. parent DNA labelled w/ N¹⁵ (heavy)

2. first replication in medium N¹⁴ (light)

3. 2nd replication in N¹⁴

• density-gradient centrifugation

origin of replication

where DNA double helix unwinds

replication forks (looks like 𝛳)

points where new polynucleotides are formed

• usually 2 bc DNA replication is bidirectional in most organisms

explain how DNA polymerase works

1. dNTP gets attacked nucleophillically by OH group on 3’ carbon of sugar

2. PPi leaves & new phosphodiester bond is formed, connecting to the following sugar’s 5’

leading strand

synthesized continuously from 5’ to 3’ at replication fork on 3’ to 5’ exposed template strand

lagging strand

• synthesized discontinuously in Okazaki fragments

• fragments linked tgt by DNA ligase

• still needs to face same way as the leading strand so it loops a bit

how many DNA polymerases in E. coli?

5

Pol I

single polypeptide chain

• repairing & patching DNA

Pol II & III

multi subunit proteins that share some common subunits

• Pol III → polymerization of the new DNA strand bc turnover # (per min) > 500,000

Pol II, IV, V

repairing enzymes

processivity

# of nucleotides incorporated before the polymerase dissociates from the template

exonuclease

enzyme that remove nucleotides from an end of RNA to correct errors made

DNA polymerase can’t catalyze de novo synthesis (from scratch), what do they need?

1. primer (10-20 bases)

   • needs the 4 ribonucleoside triphosphates

2. DNTPs

3. DNA template

4. Mg²⁺

replisomes

complex of:

• DNA polymerase

• RNA primer

• primase

• helicase

at replication fork

DNA gyrase (class II topoisomerase)

fights positive supercoils

• energy from ATP

helicase

• helix-destabilizing protein

• promotes unwinding DNA by binding at replication fork

SSB (single-strand binding protein)

stabilizes single-stranded regions by binding to them

• protects them from being destroyed bc our bodies recognize single-stranded DNA as viruses

primase

enzyme that makes short section of RNA to act as primer for DNA synthesis

primosome

complex at replication fork consisting of RNA primer, primase, & helicase

frequency of mutations

once every 10⁹–10¹⁰ (1–10 billion) base pairs

proofreading

removal of incorrect nucleotides right after they’re added to growing DNA

• reduces errors from once every 10⁴-10⁵ base pairs to 10⁹–10¹⁰

nick translation (mainly for reference only)

removal of RNA primers or DNA mistakes by Pol I using 5’–3’ exonuclease activity & filling in behind it w/ its polymerase activity