Cell phys test 2 Replication and Repair

1st step of replication of DNA

Replication forks. Allow for bidirectional replication of double stranded DNA

How is Replication Fork Formed

By DnaA protein:

-20 monomers of DnaA proteins bind to certain nucleotide sequences at origin of replication (RICH IN AT BASES)

-This ATP requiring process causes double strand DNA to melt to form localized regions of single stranded DNA

DNA helicase

Binds to ssDNA near rep fork and moves into neighboring double stranded region forcing the strands apart, unwinding the double helix

Uses atp to provide bond energy necessary to separate the hydrogen bonds

ssDNA binding proteins

keeps 2 strands of DNA separate and prevents reannealing and protects DNA from nucleases

Supercoil problem

DNA type 1 topoisomerase - cuts one strand of double helix

DNA Type 2 topoisomerase - cuts both strands of double helix, reversible

Both cut with nuclease and sealed with ligase

DNA topoisomerase I anticancer agent

camptothecin

DNA topo II anticancer agent

Etoposide

DNA polymerase reads and synthesize:

reads 3-5

synthesize 5-3

DNA pol is Mg dependent why

the 2 anionic charges on the incoming nucleotide are neutralized by it

it gets the - nucleotides to bind to the negative backbone

Leading strand is copied in what direction

in direction of advancing replication fork, continous

Lagging strand is copied in what direction

copied away from rep fork, discontinuous

small fragments of DNA are copied near replication fork

How long are okazaki fragments

100-200 nucelotides

RNA primer, why is it needed

DNA polymerase cannot initiate synthesis of the complementary DNA strand on just a single stranded template

So, it needs an RNA primer

The hydroxyl group on 3’ end of RNA primer serves as first acceptor of incoming deoxynucleotide

primer is around 10 bases long

MADE BY PRIMASE

DNA polymerases add deoxynucleotides to what end of chain

3’ end

DNA pol III

5’-3’ polymerase activity

When new nucleotide comes in, it has 3 phosphates. Two of these phosphates (PPi) are hydrolyzed (separated). This reaction drives the energy necessary to create the covalent phosphodiester bond (links nucleotides together on same chain)

DNA pol III has exonuclease activity (3’ to 5’)

DNA pol I excises RNA primer in 5’ to 3’ direction, and fills the gap in a 5’ to 3’ polymerase direction and also proofreads in 3’-5’ polymerase direction

DNA pol 1: exonuclease 3’-5’ AND 5’ to 3’, polymerase (replication)

DNA pol 2: exonuclease 3’-5’, polymerase (repair)

DNA pol 3: exonuclease 3’-5’, polymerase (replication)

DNA ligase forms

phosphodiester bond, which requires 2 high energy bonds

the high energy bonds are derived from ATP being broken into AMP and PPI (+1) and PPi being broken into two phosphates (+1)

Termination in prok occurs where

270 bp region called ter locus

ter is located 180 degrees opposite of oric

GTGTGTTGT sequence tus binds to is repeated 5x

tus protein has antihelicase activity to ensure rep of genome only completed 1x

at end of prok replication, 2 new genomes are intertwined and separated by topoisomerase II

DNA bases and nitrates

Cytosine—> uracil

Adenine—>hypoxanthine

Guanine—>xanthine

DNA glycosylase isoenzymes what do they do

snip off wrong base (U) and now theres an empty spot

AP endonucleases what do they do

snip off backbone next to empty spot that glycosylase isoenzymes snipped off, and deoxyribose phosphate lysase removes the leftover sugar-phosphate chunk

Then, DNA pol II/B patches in correct nucleotide, and ligase seals the backbone

PARP1

detects single strand breaks and then surrounds the break with ADP-ribose using NAD+ as substrate (alert for helper)

XRCC1

binds to ADP-ribose and then recruits DNA repair enzymes (helper)

How is double strand breaks repaired

homologous recombination:

uses same enzymes that normally perform genetic recombination between homologous chromosomes during meiosis

Hereditary breast cancer therapeutics

PARP1 inhibitors

leads to accumulation of SSBs in DNA, and when SSBs not repaired, theyre converted to DSBs and this leads to cells accumulating many mutations, eventually leading to cell death