Messelsohn and Stahl's Strategy for distinguishing btw old and new DNA

labeled them with isotopes by growing bateria in presense of either heavy isotope of nitrogen (¹⁵N) or light (¹⁴N)

Generation results of Messelsohn/ Stahl

generation 0 :all heavy

generation 1: intermediate

generation 2:half light, half intermed

generation 3 and on: more in light, less in intermed

Ultracentrifugation on a CsCl density gradient

origins of replication

how many in bacteria

in euks

in bacteria, one site

in eukaryotes, 10,000-100,000

E.Coli oriC

245 bps long

contain several binding sites for DnaA protein (initiate replication--also called DnaA boxes or 9-mers)

as well as AT-rich segments (help opening also called 13-mers)

DnaA proteins

recruit more of themselves; binding result in change of DNA conformation: form loops--disortion helps opening at AT rich site

ATP-dependent

DnaA structure

4 domains

domain I: N terminal: oligomerization and interaction with DnaB

domain II: flex linker

domain III: ATP binding/hydrolysis (AAA+ motif) and oligomerization

domain IV: C terminal: DNA binding (helix-turn-helix motif)

protein directionality

because of the way amino acids link up to form proteins (the repeated amide N, alpha C, and carbonyl C) there is an N terminus and C terminus

convention: N on left, C on right

AAA+ motif

domain III of DnaA protein

AAA stands for Atpases Associated with various cell Activities

common feature is coupling of energy provided by ATPase with mechanical force on a substrate--this process usually requires a conformational change of the AAA protein

DnaC

opens DnaB to fit it around DNA strand

SSBs

Single Stranded Binding Proteins

stabilize single stranded DNA prior to replication

to prevent self binding hairpin structures and keep strands seperated

no sequence specificity

cooperative binding

in eukaryotes: RPAs (heterotrimeric replication protein A)

cooperative binding

when the affinity of a ligand changes depending on amt of ligand already bound

e.g. SSBs

how many

repl forks, new strands, polymerase molecules per origin?

at least two replication forks (replication is bidirectional)

four new strands (two strands per fork)

four polymerase molecules (two molecules per fork)

DNA replication rates

(E.Coli)

size of genome: 4.6 Mb

rate of fork movement: ~1,000 bp per second

2 forks

~40 minutes to replicate the chromosome (assuming one replication event--in conditions of rapid growth, there are more than one)

DNA replication rate

(human cells)

genome size: 3,200 Mb

rate of fork movement: ~100 bp per second

10⁴-10⁵ forks

~8 hours to replicate entire genome

division rate proportional to

1. size of genome

2. time required to duplicate

DNA Pol I

ubiquitous in prokaryotes

specialized role: replaces RNA primer with DNA

only moderately processive, also involved in repair

three functional domains / enzymatic activies

1.synthesis (replace RNA with DNA)

2.proofreading (reverse exonuclease activity)

3. DNA repair-mediates nick translation (forward exonuclease activity)

Klenow fragment

large protein fragment that remains when DNA Pol I is cleaved by protease subtilisin

has 5'-3' polymerase activity

and 3'-5' exonuclease activity (proofreading)

but not 5'-3' exonuclease activity (primer removal)

makes it useful to use in lab experiments when you want to make DNA in the lab

how many DNA polymerases in E.Coli

at least five

III primary enzyme involves in replication

II, IV, V mostly involved in DNA repair

Steps leading up to prokaryotic

DNA replication

1. binding of DnaA at Origin of Replication, conformation change, melting at A-T rich region

2. DnaA recuits helicase DnaB which binds at melting site

3. DnaC also recuited along with DnaB (helicase loader)

4. SSB proteins recruited

5. helicase recruits primase (DnaG) allows polymerase to bind

Metal Ions on active site

held in placeby two aspartate residues;

metal ions: one helps 3'OH initiate nuclophilic attack on alpha phosphate and the other orients and stabilizes triphosphate of dioxyribonucleotide

1. attack by 3'OH releases pyrophosphate

2. pyrophosphate is hydrolyzed into two  phosphates (energy release drives synthesis to completion)

primer

made of RNA and is 5-10 nt long

later removed by ribonuclease (RNAseH)

RNAseH

specific for RNA/DNA hybrids

cuts out RNA primer in DNA replication

clamp loader

ATP-dependent

AAA+ motif

in E. Coli: γ-complex

in eukaryotes: RFC (Replicating Factor C)

interacts with many proteins--central scaffolding subunit at repl fork

the Τ subunit interacts directly with Pol III and DnaB helicase

Replicase

complex formed by DNA Pol III, sliding clamp, and clamp loader

Replisome

refers to entire replication machinery

all proteins on replication fork

Steps of DNA synthesis on lagging strand

1. helicase unwinds the helix

2. SSBs attach to prevent reannealing

3. primase makes short RNA primer

4. DNA Pol III (attached to sliding clamp) extend RNA primer with DNA, making short okazaki fragment

5. this process repeats forming several okazaki frags

6. DNA Pol I replaces RNA primer frags with DNA

7. ligase seals nicks

Okazaki fragments

small DNA fragments (~1,000 bp) formed on lagging strand

trombone model

lagging strand template forms loop to allow simultaneous replication of both strands

Direction of DNA synthesis

leading strand: same direction as fork

lagging strand: opposite direction as fork

synthesis on lagging strand is discontinuous and in opposite direction because DNA is exposed in 5'-3' direction

one cycle of okazaki strand synthesis

one every 1-2 seconds

Topo I

cleaves one strand of DNA duplex

(no ATP required)

catalyzes reversible nicking action

in E. Coli only removes negative supercoils

so does not relieve supercoils cause by replication

Topo II

cleaves both strands of DNA duplex

ATP required

mechanism of Topo II action

Active at replication fork

called DNA Gyrase in E. Coli

1. makes reversible covalent attachment to both DNA strands, interrupting one helix and forming a protein gate

2. gate opens and lets the other helix pass through then shuts again

3. reversal of covalent attachment restores intact double helix and two seperate helices

ciprofloxacin

antibiotic inhibits DNA gyrase

interferes with replication of anthrax

Decatenation

Topo II-like enzyme needed at end of replication

topoisomerase IV in E. Coli

untangles the two molecules of DNA once duplication is finishes

how is reinitiation of DNA replication prevented?

regulatory mechanism--uses methylation and specific sequences; way to recognize sequence just duplicated

1. in E. Coli, Dam methyltransferase catylzes methylation of GATC seqs (oriC contains many)

2. after replication newly synthesized strand is not methylated--results in hemimethylated dsDNA

3. SeqA binds to hemimethylated DNA (GATC seqs on OriC) and prevents DnaA binding

Three sources

of DNA mutation

1.copying errors introduced by DNA polymerase

2.spontaneous damage

3. environmental agents (chemical mutagens, radiation)

low mutation rate

due to what

profreading by Pol III

mismatch repair machinery

frequency of GATC seq

once every 256 bps

-mer

suffix used to specify number of nucleotides in an oligonucleotide

DNA Gyrase

Topoisomerase II in E. Coli

DNA Pol III

primary enzyme involved in replication;

requires a primer:template junction

moves continuously in 5' to 3' direction on leading strand

moves discontinuously in 5' to 3' direction on lagging strand

(E. Coli: core is a 1:1:1 heterotrimer

α polymerase

ε 3'-5' proofreading exonuclease

Θ subunit)

(In eukaryotes: Pol δ/ Pol ε)

DNA Pol I structure

resembles right hand (fingers, palm, thumb)

Thumb: maintains correct position of primer and active site

Palm: catalytic site; binds metal ions; monitors accuracy of bps

Fingers: stimulates catalysis; opens closes

Hydrolysis of pyrophosphate

mechanism of metal ions

present at active site of DNA synthesis

alters chemical environment around 3'OH of primer and around triposphates around incoming dNTP;

works on 3'OH to reduce association of O and H; produces 3'O(-) which attacks alpha phosphate of dNTP;

other one coordinates charge on beta and gamma phosphates and stabilizes the pyrophosphate

Pol I fidelity and conformational change

when correct base pair is made, one of helices in fingers domain rotates by 40°

closed form stimulates catalysis by moving incoming nucleotide closer to catalytic metal ion

Sliding clamp

increases processivity of DNA Pol III processivity increases 40 fold;

tethers DNA Pol III to template

fits behind pol III

in E. Coli called Β-clamp

forms ring around ~35Å channel in diameter--around double stranded DNA

in eukaryotes: PCNA (proliferating cell nuclear antigen)

mechanism of topo I action

nicks DNA and forms covalent DNA-phosophotyrosine bond

passes cut 3' end under other strand and reseals

types of spontaneous

DNA damage

deamination of cytosine: hydrolysis reaction of cytosine into urasil, which releases ammonia in the process

depurination:removal of purine base by hydrolysis of the beta-N glycosidic link btw the sugar and base; a hydroxyl remains in place of the base.

Repair of deamination of cytosine

uracil is removed by uracil glycosidase

abasic site recognized by enzymes, AP endonucleases,

which cut the DNA and allow DNA to repair the lesion with with new cytosine

Repair of depurination

in double stranded DNA, are efficiently repaired by Base Excision Repair (BER) pathway

in single stranded DNA undergoing replication, can lead to mutation because BER can add an incorrect base either transition mutation (addition of the incorrect purine) or transversion (substitution of a pyrimidine for the correct purine)

copying errors by Pol during replication

fidelity of DNA Pol: 1 incorrect base in every 10⁴

(in E. Coli, ~1 mutation in every 10 genes)

measured mut rate: 1 incorrect base in ever 10⁹

(in E. Coli, 10^-5 to 10^-6 mutation per gene)

Proofreading domain of Pol III: when mutation in exonuclease domain, spontaneous mut rate increases 1000 fold

nucleoside excision repair

recognizes distortions in shape of double helix

in E. Coli, UvrABCD system

RFC

replication factor C

prokaryotic correspondence: γ complex

Pol α

prokaryotic corresponding protein: DnaG

has primase and DNA polymerase activity

RPA

heterotrimeric replication protein A

prokaryotic correspondence: SSBs

uracil glycosidase

removes uracil created by deamination of cytosine

AP endonucleases

recognize abasic site left by uracil glycosidase which has removed uracil formed by cytosine deamincation

and cuts DNA to remove it

Dam methyltransferase

in E. Coli, recognizes GATC sequences and methylates the adenosine

used as way to tag newly synthesized strands (SeqAs recognize hemimethylates strands and bind to them preventing DnaA from binding)

cyclobutane ring

dimers found in DNA damaged by UV light

create covalent joining via cyclobutane ring between two adjacent pyramidines, usually two thymines

prevent replication

Replicase in Eukaryotes

Pol δ/Pol ε

PCNA

RFC

WHat does AAA⁺ stand for?

ATPases Associated with various cell Activities

Feature that allows SSBs to recruits more of themselves

cooperative binding

What two replisome proteins have AAA+ motifs?

DnaA and gamma complex (clamp loader)

Two types of Topo II

DNA Gyrase (remove pos supercoils)

Topo IV (decatentation, remove pos supercoils)

Hetero-mers

RPAs (trimeric)

MCM (hexameric)

DNA Pol III (trimer)

Ku of NHEJ pathway (dimeric)

UvrA (dimer)

What does NHEJ stand for?

Non homologous end joining repair pathway

(double strand breaks)

Hydrolysis of ATP

DnaB

a helicase; hexameric ring six identical subunits, that encircles single strand; uses ATP hydrolysis to seperate strands; also has ATPase domain

unwinds DNA in advance of replication fork

when DnaB initially binds to DnaA, it is associated with DnaC which helps load DnaB onto DNA strand; recuits DnaG (primase/RNA polymerase) and interacts with it

in eukaryotes: (MCM complex--heterohexameric)

one helicase and loader per fork

exonuclease activity

Pol I

3'-->5' activity

proofreads newly synthesized DNA

exonuclease domain 30Å from active site

incorp of incorrect base pair causes melting of 4-5 nucleotides

exonuclease removes last nucleotide

mismatches

how do they arise

can arise from Pol III error

or deamination followed by replication

MutH

has dGATC endonuclease but only in course of methyl-directed mimatch repair activation by MutS, MutL and ATP

DNA polymerase

can only add free nucleotides onto 3' end of newly growing chain --this is why DNA synthesis only goes in 5'-3' direction

uses a magnesium ion for its catalytic activity

can only add onto preexisting 3' OH group (needs a primer)

primase

specialized RNA polymerase

generates the primer used for replication

called DnaG in E.Coli

active site also contains metal zinc

recuited by helicase DnaB (direct prot-prot interaction)

in eukaryotes: Pol α (primase & polymerase activity)

MutHLS mismatch repair

excision repair

two mechanisms

replaces chemically modified bases

such as bases modified by alkylation (methylation), oxidizing agents, radiation

1.base excision repair

2. nucleotide excision repair

Thymine-Thymine dimers

specific type of more general occurance pyrimidine dimer

covalent bonding of two adjacent thymines often catalyzed by UV radiation or chemical mutagenic agents; create cyclobutane ring

DNA repair systems recognize large kinks in backbone. repaired by nucleotide excision repair

UvrABCD repair system

system for nucleotide excision repair in E. coli

1. UvrA dimer and UvrB form complex (ATP dependent step); 2. bind to DNA (ATP dependent step); 3. translocate along strands until distortion is found (ATP dependent step); 3. conformational change provokes bending and melting at spot (ATP dependent step); 4. UvrA dissociates (ATP dependent step) recruits UvrC which binds; 5. UvrC endonuclease activity makes dual incision on one strand (ATP dependent step) 6. UvrD helicase releases fragment 7. Pol I and ligase fill and seal gap

double strand breaks

two repair mechs

caused by γ radiation and X ray

most dentrimental DNA lesion

net result: deletion

1.DSB repair pathway (recombination)

2. non homologous end joining (NHEJ) pathway

NHEJ found in Eukaryotes and bacteria

NHEJ repair pathway

1. 2 Ku heterodimer proteins attach to both ends of DSB, recruit protein kinase

2. Ku dimers interact with each other and form a synapse (bring two ends closer together)

3. Ku helicase seperates strands of two ends

4. micropairing of 1-2 bps of one 3' end to another 3' end

5. remaining unpaired 5' ends removed by nuclease

6. ligase reseals two ends

PCNA

proliferating cell nuclear antigen

prokaryotic corresponding protein: B clamp

base excision repair

glycosylase removes base by hydrolyzing glycosidic bond