DNA Replication

Meier-Gorlin Syndrome

MCM5 gene has a mutation which impairs helicase function, which leads to inefficient DNA replication initiation. This reduces cell proliferation during development resulting in primordial dwarfism and skeletal abnormalities.

Conservative replications yields

an original intact DNA molecule and one entirely newly synthesized DNA

Semi-conservative replication yields

two DNA molecules, each with one parental and one newly synthesized strand

Dispersive replication yields

two DNA molecules that are hybrids of parental and newly synthesized DNA

Significance of Meselson and Stahl experiment

Found that DNA replication is semiconservative

DNA polymerase replicates

DNA

What are the substrates for DNA polymerase

dNTPs

DNA polymerase catalyzes the extension of a DNA strand one ________ at a time

dNMP

What direction does DNA polymerase synthesize

5’ → 3’ direction

DNA polymerase requires

a template and DNA or RNA primer

DNA synthesis requires two ____ at active site

Mg2+

An error in replication can introduce a _______ into the genome

mutation

DNA Pol active site restricts base paring to 

Watson-Crick-Franklin bp (A-T and G-C)

Mutation rates is typically very 

low

DNase is specific for

DNA

RNase is specific for

RNA

An enzyme that removes bases from DNA is called a

nuclease

Exonuclease breaks

a phosphodiester bond at one end of a polynucleotide chain.

Endonuclease breaks

a phosphodiester bond within a polynucleotide chain

Characteristics of endonucleases

• sequence independent or specific

• single-strand nick or double-strand break

Excinuclease breaks

two phosphodiester bonds within a single polynucleotide chain

Restriction endonucleases/enzymes

types of endonuclease that only break phosphodiester bonds at a specific DNA sequence (restriction sites)

Restriction sites are typically…

short, palindromic (same sequence read 5’ → 3’)

High-fidelity DNA polymerases have two active sites:

1. a catalytic site for DNA synthesis

2. 3’ → 5’ exonuclease site for removing mis-incorporated nucleotides

DNA synthesis begins at

the origin of replication sequence

Bidirectional synthesis definition

DNA is synthesized in both directions starting at the origin

DNA is synthesized by DNA Pol at sites called

replication forks

A replication fork is where

parent DNA is being used as a template for replication by DNA polymerase

Helicase function

unwinds parent DNA

Leading strand’s DNA synthesis occurs

continuously 5’ → 3’

Lagging strand DNA synthesis occurs 

discontinuously in Okazaki fragments as a series of 5’ → 3’ reactions

Formation of supercoils occurs due to

under-winding or overwinding DNA causes torsional stress

Topoisomerases are the enzymes that

add/remove supercoils in DNA by cutting phosphodiester bonds in one or both strands, unwrap the helix, and then reseal the strands

Topoisomerase II or DNA gyrase in bacteria introduces

negative supercoils to compact the genome

Fluroquinolones are antibiotics that target

bacterial DNA gyrase

Characteristics of fluoroquinolones

• block ability to reseal DNA → double-strand breaks

• selective toxicity for bacterial enzymes

What is the regulated (rate-limiting) step in the control of DNA synthesis?

Initiation

DNA unwinding element (DUE) is

an AT rich segment where strand separation occurs

The first protein that comes in for DNA replication at the ORI

DnaA

Dna binds to _____ to become active

ATP

DNAC binds _____ and loads ______________ at both ends of the replication bubble

ATP; DnaB helicase

oriC DNA is methylated by

DNA adenine methylation (DAM) methylase

After DNA replication, DNA is __________

hemi methylated

Hemi methylated means

One strand is methylated and one strand is not

Does DNA polymerase I and III have 3’ → 5’ exonuclease (proofreading) activity?

Yes

Does DNA polymerase I and III have 5’ → 5’ exonuclease activity?

ONLY DNA polymerase I

Compare polymerization rate and processivity of DNA polymerase I and III

DNA pol III has higher rates and processivity

Core polymerase (Pol III) catalyzes

DNA synthesis

The clamp loader serves as

• a scaffold for DNA polymerase III complex

• assemble the beta clamp onto DNA using ATP

• coordinates the replication fork by interacting with DnaB helicase through T subunits

Beta clamp (sliding clamp) tethers

core polymerase to DNA

Beta clamp increases processivity by

decreasing polymerase dissociation from DNA

Primase is a ____ polymerase

RNA

DNA polymerase requires a primed DNA template that has a pre-existing

3’ - OH

Characteristics of primase

• DNA template-dependent

• primer-independent

• Synthesizes a <9 nt RNA primer at the beginning of the leading strand and each Okazaki fragment

Function of DNA gryase at fork

removes + supercoils ahead of fork

Single-strand binding protein (SSB) protects

single-stranded DNA from nucleases, reannealing, and secondary structures

Lagging strand synthesis steps

1. DNA helicase travels along lagging template strand in 5’ → 3’ direction and unwinds DNA

   1. DnaG primase occasionally associates with DnaB helicase and synthesizes a short RNA polymer

   2. Primase functions at the replication9 fork as core polymerase nears completion of an Okazaki fragment

2. New beta clamp loaded onto the lagging strand at each new RNA primer by the clamp loader

   1. clamp loader binds ATP then sliding clamp

   2. Clamp loader opens the sliding clamp at one subunit itnerface

   3. ATP hydrolysis closes the clamp around the DNA and the clamp loader dissociates

3. DNA pol III synthesis of an Okazaki fragment is complete when it reaches the previous primers

   1. lagging strand core pol pauses and releases beta clamp

   2. lagging strand core pol transfers to newly loaded beta clamp

   3. old clamp is left behind

4. DNA Pol I enters and uses 5’ → 3’ exonuclease to remove primer and synthesizes DNA to fill in gap

5. DNA ligase repairs nick linking Okazaki fragments into a single DNA strand

Function of DNA ligase

links two existing DNA chains together by forming a phosphodiester linkage. Joined ends result in a continuous 5’→3’ DNA strand

Bidirectional, semi-conservative replication yields

two DNA molecules with identical nucleotide sequences

Catenated state

after replication, circular chromosomes are linked together like links in a chain

Function of topoisomerase IV

Separation of catenated circles in E. coli

Do eukaryotes have one or multiple origins of replication?

multiple

S phase

DNA synthesis doubles the amount of DNA in the cell. RNA and protein are also synthesized

Origin of replication complexes (ORC) bind tightly to DNA in ____ phase

G1

CDC6 and CDT1 join and load

helicase mini chromosome maintenance (MCM)

In S phase, replication is initiated by phosphorylation of complex proteins by a

cyclin-dependent kinases (CDKs)

In G1 phase, CDKs are ______, licensing is ________, and origin firing ________

off; allowed; prevented

In S phase, CDKs are ______, licensing is ________, and origin firing ________

on; prevented; allowed

In eukaryotes, what is the helicase

MCM

In eukaryotes, what synthesizes the leading strand

DNA pol E

In eukaryotes, what synthesizes the lagging strand

DNA pol Delta

In eukaryotes, DNA Pol alpha-primase is

a complex that contains primase for RNA primer synthesis and a separate DNA synthesis actvity

In eukaryotes, what is the clamp loader

Replication factor C (RFC)

In eukaryotes, what is the sliding clamp?

Proliferating cell nuclear antigen (PCNA)

In eukaryotes, what is the ss DNA binding protein (SSB)?

Replication protein A (RPA)

DNA Pol D makes the lagging strand and allows for

strand displacement synthesis

In eukaryotes, what clips off the overhang after strand displacement synthesis

Flap endonuclease-1 (FEN1)

In eukaryotes, what repairs the nick left in the sugar-phosphate backbone to join the two Okazaki fragments

DNA ligase

Telomeres

DNA structures at the ends of eukaryotic chromosomes that protect the chromosome

Characteristics of telomeres

• consist of repetitive short sequence

• TTAGGG - TG strand

• TG strand is longer than complementary strand (CA strand)

• No important genetic information

T loops in telomeres

specialized structure that sequesters the single-stranded end of the telomere by base pairing which protects 3’ ends of chromosomes from nucleases and enzymes that repair ds breaks

Shelterin proteins protect

ss 3’ end within a DNA duplex

When telomeres reach a critical length, cells can no longer divide and will…

enter G0 permanently (senescence) or apoptosis

Telomerase is a specialized

reverse transcriptase

During telomere synthesis, the complementary (CA) strand is synthesized by 

DNA Pol alpha-primase

During telomere synthesis, following the removal of the RNA primer, the overhanging 3’ end base pairs to the CA strand, forming a….

T-loop

What cells can make telomerase?

Sperm, eggs, and embryonic stem cells

Reverse transcriptase

RNA → DNA