MBB 331 Lecture 9

What is a DNA nick

A break in only one DNA strand

Why is a nick usually not very dangerous

intact strand is used as a template and ligase can rejoin the break

What is a double-strand break (DSB)

Both DNA strands are broken

Why are DSBs dangerous

no intact complementary strand at the break site chromosomes can be lost or rearranged

What are the two major ways to repair DSBs

Homologous recombination and NHEJ

What is a DNA leison

when have things like dimers/alkyls/methyl groups attatched

Outcome #1 of a damaged template

Translesion synthesis (TLS). Replication continues over the lesion

Double strand break causes what

fork to collapse and broken chromosome

Collapse of the fork triggers what

recombination repair process

DSB Repair requires what

undamaged source of duplicative genetic info

repairing a DSB: first step?

Broken ends are processed to generate 3' single stranded extensions.

repairing a DSB: second step?

3' ss ends are used for strand invasion.

Repairing a DSB: third step?

Replicative extension of invading strand.

Outcome #2 of a damaged template

Fork collapse caused by a single-strand break, producing a DSB

Outcome #3 of a damaged template

Replication stalls until repair occurs

Replication stall description

start the repair and aren't able to finish it by the time replication fork comes along, leads to collapse

step one in replication stall

1. theres is a lesion and theres one strand that has lesion and replication stalls

step two in replication stall

fork regression so we backtrack and zip it up

step three in replication stall

strands are now complementary and base pair creating 3' OH

step four in replication stall

Polymerase can come and extend

step five in replication stall

branch migration and we can unzip

What is the second alternative in the pathway in replication stall

OR We can repair lesion and then digest nuclease

Outcome #4 of a damaged template

Lesion bypass. Replication restarts downstream and leaves a gap behind

SDSA Step One

end processing to create 3’ ss ends

SDSA Step Two

Strand invasion- the strand will go up into the gap

SDSA Step Three

Branch Migration (2 holliday junctions)

extend through the gap

SDSA Step four

strands dissociate and anneal together

What is SDSA used for

Repairing DSBs using homologous DNA without forming a crossover

SDSA step five

replication and ligation of gaps

What happens after the break

Ends are processed to create 3' ssDNA tails

Which protein performs strand invasion

Recombinase (RecA in bacteria)

What happens after strand invasion

DNA polymerase extends the invading strand

How is SDSA completed

Helicase displaces invading strand, strands extend, DNA is ligated

Does SDSA cause crossover

No

Cutting vertically at the X's causes

no crossover ends are the same colours

Cutting horizontally at the Y's causes

crossover ends are different colours

What is homologous recombination

Repair of DSBs using homologous DNA as a template

What protein complex processes DSBs in bacteria

RecBCD

What does RecBCD do

Generates 3' ssDNA tails and loads RecA

What are RecBCD

two helicase domains ones 3'-5' and the other is 5'-3' have different speeds

RecD speed

5-3 fast moving

RecB speed

3-5 slow moving

RecB nuclease

degrades both strand

When do we digest DNA till

the chi site

The chi site is bound by what

Rec C preventing further degradation of 3' end

What is the central recombinase

RecA

What does RecA do

Finds homology and promotes strand invasion

What structure forms after strand invasion

D-loop and eventually Holliday junctions

What proteins promote branch migration

RuvA and RuvB.

What protein resolves Holliday junctions

RuvC

When is NHEJ commonly used

G1 phase when no sister chromatid is available

Is NHEJ accurate

No, it is mutagenic

Which proteins first bind broken DNA ends

Ku70 and Ku80

NHEJ Which proteins are recruited next

DNA-PKs and Artemis

What does Artemis do

Processes damaged ends using nuclease activity

Which enzyme seals the DNA ends

Ligase IV complex.

NHEJ Step One

recognize the double stranded break and proteins come in and widen break

NHEJ Step Two

creates tiny overhangs and paste them together

NHEJ Step Three

extends and ligates the ends with ligase IV

Are DSBs always accidental

No. During meiosis they are intentionally created

Why are DSBs generated during meiosis

to promote homologous recombination and genetic diversity

What is a gel mobility shift assay used for

Detecting protein-DNA binding

Why does DNA move slower when bound to protein

the DNA-protein complex is larger than free DNA

What does a shifted band indicate

Protein is bound to DNA

What can EMSA tell you

Whether binding occurs

What can EMSA NOT tell you

The exact binding location

When do we see a supershift

when both proteins are attatched

double strand break repair (DSBR)

one pathway of DSB to complete repair

holliday junctions are cut by endonucleases

HJ resolution

extension and ligation of gaps

DSBR step one

end processing to generate 3’ ss ends

DSBR step two

strand invasion (2 holliday junctions)

DSBR step three

branch migration

DSBR step four

holliday junctions cut by endonucleases (HJ resolvases)

DSBR step five

filling in and sealing gaps