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Summary of last lecture

• Restriction enzymes scissors for precisely cutting DNA

• Restriction maps can be generated by cutting a piece of DNA with

different restriction enzymes in double digests
 Genome Maintenance

DNA replication, Damage & Repair, Modification,

Recombination, Gene conversion
DNA Replication
 Cell cycle

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Prophase Nucleus and cell divide
Metaphase
Anaphase
Telophase
M phase

G2 phase G1 phase
Interphase

S phase
DNA replication

DNA replication occurs prior to mitosis (however limited synthesis may occur
during recombination or for repair of damaged DNA)
 Important Concepts

• Lagging strand and Leading strand

• Semiconservative and Dispersive replication
• Origins of replications (replicons)
• Replication is basically formation of phosphodiester bonds
• Replication forks
• Replisome (complex of different proteins for replication)
• Enzymes and Proteins
• Errors in replication
• Proofreading
• Direction of polymerization
• Functional groups involved in bond formation
• Topoisomerase (Gyrase)
 DNA replication is semiconservative

N15 labeled DNA Starting DNA is heavy (same high

(heavy) density)

Replication in presence After first replication DNA

Of N14 Is hybrid (medium density)

Second round of replication in presence of N14 yields 2 DNA molecules

which are light and 2 molecules which are hybrids (medium density and low density)

Meselson and Stahl experiment with N15

 DNA replication

• Initiation involves recognition of the position(s) on a DNA molecule

where replication will begin

• Elongation concerns the events occurring at the replication fork,

where the parent polynucleotides are copied

• Termination is only vaguely understood, occurs when the parent

molecule has been completely replicated
 Origins
• Synthesis begins at origin of replication (Replicons)

• Usually AT rich regions (melt quickly)

• DNA sequences recognized by replication initiator proteins

• In bacteria, one origin of replication for one chromosome

• OriC is a 9 base-pair (bp) sequence that is repeated four times and
three A–T rich 13 bp repeats

• In eukaryotes multiple origins of replications on each chromosome

 Replication forks

• As DNA unwinds for replication, polymerase start synthesis of DNA

on both DNA strands in both directions forming replication forks

• Replication forks are ALWAYS formed in pairs

• From one origin, there will be two forks

• The two forks together form a replication bubble as they move in

opposite directions away from a common point of origin

• Thus you can say that DNA replication is bidirectional

Origins and Replication forks
3
5
 Synthesis
• Helicase opens the strands (breaks hydrogen bonds)

• Primase first synthesizes a new primer which is about 10

nucleotides in length (RNA primer) (Primosome)

• SSB Single strand binding protein (SSB) binds single-stranded DNA

at the replication fork and physically blocks potential re-hybridization
(RPA in eukaryotes)

• DNA polymerase extends the RNA primer (5’-3’)

• Leading and lagging strands (Okazaki fragments)

• The distance between two primers on the lagging strand is about

1000-2000 nucleotides in bacteria, and about 100-200 nucleotides
in eukaryotic cells (lead to different Okazaki fragment lengths)
One replication fork
DNA pol III is the main replicating enzyme in
bacteria
RNA primers have to be removed and replaced by
DNA. This is done by DNA pol I in bacteria
DNA ligase - seals nicks in DNA by linking up 3'
hydroxyl groups (acceptor with adjacent
5' phosphate groups (donors)-forms
phosphodiester bonds
 Movie

http://www.dnatube.com/video/335/Animated-DNA-Replication
 DNA polymerases
• RNA polymerase can synthesize a new strand whereas the DNA
polymerase can only extend an existing strand (works on partially
double stranded DNA)

• Needs a DNA with base-paired 3′ nucleotide with free OH group

• 5’-3’ is the polymerase activity (with respect to direction of DNA)

• 5’-3’ exonuclease activity is also there which is used for removing

RNA primers (exhibited by Polymerase I)

• 3’-5’ exonuclease activity removes mismatched nucleotides

• There are separate DNA polymerase for leading and lagging

strands in eukaryotes
• Many enzymes and subunits of DNA polymerase

• DNA Polymerase III makes 1 in 109 or 1011 mistakes or errors

• We have discussed mutations which can be produced due to
replication errors
 FEN1

FEN1 (Flap endonuclease 1) removes RNA primers in eukaryotes

Endonuclease

Also has 5’-3’ exonuclease activity for nicked or gapped ds-DNA

Also has RNAse H activity

It is part of DNA polymerase complex

 Torsion problem

• Twists or knots can be produced in DNA as it is helical (like spring)

• Topoisomerase prevents tangling of DNA by binding at different
sites and
• Cutting DNA

• DNA isomerase which change topology (structure)

• Change linking number (number of time two helical molecules wind
around each other)
• Help in unwinding the helix ahead of the fork without the molecule
having to rotate
 Topoisomerase I

• Topoisomerase I produces a nick (break) in one strand of DNA

and reseals it to relieve torsion (breaks a phosphodiester bond)

• Reaction is reversible, so after the strain has been released, the

nick is sealed (Does not need Ligase)

• Favorable reaction, no ATP is required

 Topoisomerase II

• Topoisomerase II cuts both backbones of double stranded DNA

• Requires ATP

• One double-stranded DNA can now pass through another

• Removes knots and tangles that can form within and between DNA
molecules

• Separate daughter strands ( topo II in eukaryotes and by topo IV in

prokaryotes)
• Topoisomerase forms scaffold-like network that permeates the
nucleus

• Helps in maintaining chromatin structure

• Helps in unlinking DNA molecules during chromosome division

• Most topoisomerases are only able to relax DNA

• However prokaryotic Type II enzymes, such as the bacterial DNA

gyrase and the archaeal reverse gyrase, can carry out the reverse
reaction and introduce supercoils into DNA molecules
 Telomeres
• Telomeres can be thought as equivalent to the plastic or metal
sleeves at the end of shoelaces called "aglets"
• Just as the ends of shoelaces keep them from fraying, telomeres
keep DNA from unraveling or fusing

• Eukaryotes add telomeric sequences to the ends of their DNA

(ends of chromosomes)

• Telomerase adds non-coding, redundant sequences to the ends of

the DNA (This is heterochromatic)

• This simply makes the overhang many-fold longer

• Primase then lays down an RNA primer at the extreme end of this
new structure which is then replicated by a DNA polymerase
5’ 3’
3’ 5’

GAP
 Telomerase

• Telomerase has a reverse transcriptase activity

• Telomerase has a RNA molecule (so it is a riboprotein)
• The RNA molecule has sequences complementary to telomeric
DNA

• TERF2 in humans, recognize telomere sequences and bind to

protect ends of the chromosomes

• In vertebrates telomerase activity is present in germ and stem cells

and telomerase activity is usually not found in somatic tissues

Medical relevance
One factor in aging on cellular level
Senescence
Some cancer cells have high telomerase activity
 Telomeres and longevity
• Telomeres shorten as we age, but the telomeres of centenarians are
remarkably long, more like those of people three decades younger
or even younger

• Each time a cell divides, some of the telomere is lost; when it

becomes too short, the cell dies.

• Healthy people with longer telomeres seem to be at lower risk of

age-related illness

• Telomere diagnostics and TeloYears test, measure telomere

length

• Public testing is premature

• http://www2.macleans.ca/2013/05/14/how-long-will-you-live/
Variations of semiconservative
replication
 Displacement Replication

• Involves continuous copying of one strand of the DNA helix

• The second strand is displaced and is subsequently copied after

synthesis of the first daughter genome has been completed

• Human mitochondrial DNA

 Rolling Circle Replication

• Is used by l and other bacteriophages, rarely in bacterial plasmids

• Initiates at a nick which is made in one of the DNA strand
• The free 3′ end that results is extended, displacing the 5′ end of
the same DNA strand

• A strand rolling off the circle

• There is no termination point so synthesis often produces
concatamers (a series of linked chains) of several circle lengths (all
single stranded which are made double stranded later)

• As the synthesis proceeds, the other end of the template strand is

displaced from the double-stranded circle and then copied
 Important things to remember
• DNA polymerase always needs a primer for de novo synthesis
• The primer should always have a free 3’ OH group
• The incoming nucleotide should always have a triphosphate at the 5’
end
• DNA will only be polymerized in 5’-3’ direction
All DNA polymerases are 5->3 in their polymerizing ability

Details worked out in E. coli DNA Polymerase I is the most abundant

enzyme for replication but acts more in removing the RNA primers,
filling in the resulting Gaps (Cutting, Patching up and mending)

• DNA polymerase III acts as the major replicating enzyme

• Many accessory proteins are needed
• Helicase (breaks hydrogen bonds), DNA ligase (makes
phosphodiester bonds), Topoisomerase (breaks phosphodiester
bonds)