Lecture 3 - DNA Replication

Stahl’s Experiment

e. coli is grown in a heavy nitrogen isotope so that its DNA becomes fully labelled with N15 then transferred to N14 (lighter isotope)

samples are collected after 1 and 2 generations of cell division

DNA was extracted and analyzed → found a shift in distribution of DNA weight to lighter side which means DNA is semi-conservative

Semi-Conservative

DNA replication is semi-conservative - newly replicated DNA has a new strand and an original strand

replication is bidirectional, both strands copied at the same time

Minimum Requirements for DNA Replication

• original DNA strand - template

• enzyme to polymerize the nucleotides into a strand of DNA - DNA polymerase

Problems with DNA Replication

1. DNA is a helix - solution is to unwind by helicase

2. template has antiparallel strands and polymerase can only catalyze in the 5’3 direction between OH and phosphate, respectively - solution is semidiscontinuous replication

3. DNA polymerases cannot initiate chain synthesis de novo - solution is to use a primer made by primase to initiate synthesis

Semidiscontinuous Replication

both strands are synthesized from 5’-3’, one continuously and the other in discontinuous fragments

3’-5’ leading strand is replicated continuously

5’-3’ lagging strand is replicated in short fragments called Okazaki fragments

Replisome

multicomponent machine comprised of a helicase, primase, DNA polymerase, and 3’-5’ exonuclease

Intro to Bacterial DNA Replication

circular genome has a single origin of replication (OriC) and 10 termination sites

OriC is AT rich and termination site has 10 copies of 23 bp sequence specific replication arrest site, bound by Tus

bacteria have 5 DNA polymerases but not all are involved in replication

Bacterial Replication

1. DnaA (initiator) binds to OriC and melts the DNA

2. DnaB (helicase) encircles lagging strand and uses ATP hydrolysis to unwind the dsDNA

3. ssDNA is bound by SSBs to prevent breakdown by nucleases

4. primase, DnaG makes a 10-12 nucleotide RNA primer using DNA as a template

   1. DnaG interacts with DnaB meaning primers can only be made at replication forks

5. sliding clamp sliding clamps beta are loaded onto the DNA by clamp loader. Clamp loader uses ATP
   hydrolysis to load beta onto the DNA

6. DNA pol III are tethered to clamp loaded and also to DNA

   1. 2 of 3 pol III molecules are placed on lagging strand and take turns extending RNA primers into Okazaki fragments

7. replication fork advances and makes a DNA loop

8. Pol III is released after Okazaki fragment is made or by collision with previous fragment

9. new sliding clamp assembles on another RNA
   primer, and DNA polymerases are associated
   anew to make the next Okazaki fragment

10. synthesis continues until polymerases meet each other at the Ter sites

11. after lagging strand is done pol I removes ribonucleotides of primer and replaces with complementary dNTPs

12. DNA ligase catalyzes formation of phosphodiester linkage between adjacent fragments

To Be Resolved After Bacterial Replication

• removal of RNA primer

• ligation of the DNA fragments

• induction of supercoiling

DNA Polymerase I 

made of 2 subunits:

1. subunit with 5’-3’ polymerase activity and 5’-3’ exonuclease activity called Klenow Fragment

2. subunit with 3’-5’ proofreading activity

Resolving Bacterial Supercoil

as replication fork moves forward, positive supercoiling happens ahead of the fork

freshly replicated strands are becoming negatively supercoiled

torsional strain is relieved by topoisomerase:

1. type I does not require ATP hydrolysis

2. type II requires ATP hydrolysis

DNA relaxed by topoisomerase can be visualized on agarose gel → supercoiled DNA will move faster through the gel since it’s tightly packed

Type I Topoisomerase

works independent of ATP to unwind DNA

bacterial topoisomerase I can only relax negatively supercoiled DNA but eukaryotic can relax both negative and positive supercoils

in bacteria: binds negatively coiled circular DNA and unwinds DNA by making a transient single strand break in the phosphodiester bond but doesn’t make free ends

• enzyme can then pass the unbroken ends between the cut strand and religates the DNA cut strand

Catenation

interlocking of circles of DNA like links in a chain

topoisomerase type II can unknot or decatenate entangled DNA molecules

Type II Topoisomerase

ATP dependent and can relax both positive and negatively supercoiled DNA; decatenate and unknot DNA

makes transient double strand breaks

• can induce negative supercoils

• makes a dimer when ATP binds, 2 ATP molecules are hydrolyzed to allow for rotation of the gyrase for cleavage and subsequent rotation for reset

Eukaryotic Replisome

linear genome with bidirectional replication means the ends are a problem

• there are multiple origins of replication called origins (50000-80000 in humans)

• yeast have autonomous replicating sequence but mammals don’t have a conserved consensus sequence

• consensus sequence: ideal form of a DNA sequence found in slightly different forms in different organisms'

replication occurs in replication factories in the nucleus - more than 40 active forks clustered here in pulse chase experiments

The rate of genome replication in eukaryotes is affected by ________ and ________.

the number of origins used; the rate at which the origins are initiated

Active Replication Fork

synthesis is restricted to the S phase of the cell cycle

origin recognition is different from replication licensing: regulated formation and activation of prerreplication complexes by CDK to make sure DNA only replicates once per cycle

1. chromatin must be opened up and histones removed

2. origin recognition complex (ORC) and Cdc6 binds the origin and recruits a single Mcm2-7 helicase in a complex with Cdt1

3. other proteins like Cdc45 and GINS are loaded and completes pre-initiation complex

4. Cdc45, Mcm2-7, GINs are transformed into an active helicase called CMG that encircles the leading strands

Replication Licensing

Mcm2-7 acts as the licensing protein and a helicase in the CMG complex

• ATP hydrolysis by ORC with the help of Cdc6 are needed to load mcm2-7 at the origin

• mcm2-7 is only loaded on the origin when CDK activity is low

• when CDK activity is high the licensed origins fire and CDK can phosphorylate Mcm2-7 and prevent it from being loaded at the origin

• CMG helicase complex goes with replication fork and unwinds DNA ahead of the fork powered by ATP hydrolysis

• ssDNA is bound by RPA as DNA is unwound which protects DNA from nucleases

  • positive supercoil ahead of fork and negative supercoil behind the fork need to be resolved by topoisomerase

Elongation in the Eukaryotic Replisome

replication involves polymerase switching

once CMG helicase separated DNA, RNA primer has to be laid down

• DNA pol-alpha/primase binds to initiation complex and makes a primer of 7-10 nucleotides of RNA and then 25-35 nucleotides of DNA → DNA part serves as initiator

• DNA pol-alpha/primase switches to lagging strand for DNA pol-delta for replication; DNA pol-epsilon does replication on the leading strand (polymerase switching)

Elongation and Proofreading

PCNA: proliferating cell nuclear antigen is central to DNA pol switching

• this is the sliding clamp for the eukaryotic replisome - gets loaded onto duplex DNA by clamp loader Replication Factor C (RPC) by ATP hydrolysis

PCNA works to prevent the DNA polymerase from losing its place at the replication fork and holds DNA complex together in front of the fork

Proofreading and Termination

DNA polymerases are high fidelity enzymes and proofreading 3’-5’ exonucleases in Pole (leading) and Pold (lagging)

abnormal geometry from mismatched base pair → steric hindrance that pauses pol and the exonuclease domain can excise the mismatched nucleotide before polymerase resuming elongation (slows down overall replication)

Termination and Maturation

replication terminates when the replisomes from neighbouring origins converge

• after termination, RNA primer must be removed, gaps filled in, Okazaki fragments joined

• when DNA Pol-delta reaches the 5’ end of the preceding Okazaki fragment → strand displacement

• flap endonuclease 1 removes the 5’ RNA flap

unreplicated DNA gaps are filled in by pol-delta on the lagging strand → double nick

• ligase I joins the ends and distinguishes between RNA with substrates and dNTP is filled in based on the shape of the DNA

PCNA is needed for Okazaki fragment maturation and interacts with ligase I, FEN-1 and pol-delta

nucleosomes that were disassembled into H3-H4 tetramers and H2A-H2B dimers for replication reform behind the replication fork → histones are deposited as soon as there is enough DNA to reform nucleosomes

End Replication Problem

removing the final RNA primer from the lagging strand would leave an unreplicated portion of the DNA but there is no more DNA upstream for pol to fill the gap

• left uncorrected, our DNA would get shorter after every round of division → chromosomal instability, loss of genetic information, cell death

Telomeres

telomeric DNA has arrays of TTAGGG repeats which makes a region of dsDNA ending in a single stranded G-overhang

telomeres and shelterin protein complex protect the ends of chromosomes preventing chromosomal end fusions and preventing the ends from being recognized as double strand breaks

Identifying Telomerase

using cell-free extracts from tetrahymena thermophilia since they have a lot of telomeres

• incubated with an oligonucleotide containing 4 repeats of TTGGGG (at end of chromosomes)

  • sequence was known but protein making telomeres was unknown

• provided nucleotides (dNTPs radiolabelled or unlabelled)

• after incubation, separated the products of the reaction by gel electrophoresis

Telomerase

ribonucleoprotein complex has an RNA subunit and a protein subunit

protein component: reverse transcriptase called telomerase reverse transcriptase (TER)

• reverse transcriptases make DNA from RNA

RNA subunit is a non-coding RNA called telomerase RNA component (TERC) or telomerase RNA (TER)

A critical feature of the RNA subunit of telomerase is a _____ structure.

pseudoknot

Cajal Bodies

non-membrane bound nuclear suborganelles made of proteins and RNA

plays a role in transcription and RNA processing

this is how telomerase exists when not copying telomeres

Telomerase Recruitment

250 copies of telomerase in each cell traverse the nucleus at 4 uM/s for telomeres

once a telomere is found, telomerase binds and is ready to go

Maintenance of Telomeres

telomerase RNA has a region of complementarity to the C strand so the RNA can then serve as a template for telomere repeat synthesis

telomerase extends the template for the lagging strand (adds nucleotides 5’-3’ on the 3’ overhanging strand)

• Undergoes repeated translocation and elongation to create tandem arrays

• results in the either the lagging strand
  synthesis or t-loop formation

The C strand fill-in, in yeast and ciliate, is carried
out by the replication machinery including DNA pol
α/primase, DNA polymerase δ, RFC, and PCNA

C strand

shortened 5’ strand at the completion of the lagging strand

G strand

3’ 12-16 nucleotide long overhang on the leading strand

Regulation of Telomerase

activity must be regulated to prevent telomeres from getting too long or too short

• shelterin: 6 proteins important for protecting chromosome ends and may play a role in preventing telomerase access to telomeres by making a folded structure

• t-loop: invasion of double stranded telomeric DNA by the single stranded G strand

• g-quadruplex: can also block telomerase activity

if there is an overly long telomere, limiting abundance of shelterin lets TZAP bind long telomeric repeats excise the T-loop

Telomere Maintenance in Drosophila

no telomerase activity → ends are maintained by addition of retrotransposons to ends

Alternative Lengthening of Telomeres (ALT)

another method of maintaining telomeres 

observed in telomerase negative tumours where telomerase isn’t used

double stranded breaks at the telomeres triggers “break induced telomere synthesis”

Cells and tissues differ in the amount of _____ activity.

telomerase; specialized cells have less telomerase activity, highly repetitive cells have more telomerase activity

• potential consequence in progressive shortening of telomeres by Hayflick Limit (limits # of doublings a somatic cell can do)

  • when cells reach their limit they enter an irreversible state of cellular aging called senescence and cells no longer divide

Telomerase in Aging and Cancer

• length of telomeres can act as molecular clock where shortening can trigger aging

• telomerase reactivation is considered a fountain of youth

• most cancer cells have reactivated telomerase and others may use ALT pathway to maintain the telomere lengths

• effect of experimental activation of telomerase on normal human somatic cells (telomerase reactivated

Rolling Circle DNA Replication

extrachromosomal DNA in bacteria has to be replicated (plasmids) 

gene amplification events also use this

1. dsDNA is nicked on the positive strand by endonuclease

2. free 3’ end of nicked strand = primer for host polymerase to start replicating leading strand with negative strand as the template

3. 5’ end of positive (outer) strand is displaced as negative strand gets copied

4. unit length of new positive strand is made and the displaced parental parent strand is cleaved off and ligated

5. replication continues and makes another positive strand with negative template

6. many ssDNA are made that can be a template for lagging strand synthesis and needs a primer

Rolling circle DNA replication is _____ and needs 2 separate steps for ______ and _____ strand synthesis.

asymmetric; leading; lagging