11/17/2015

The Flow of Biological Information

Fundamental Genetics
Lecture 10

DNA Replication
and Synthesis

Replication
DNA

Transcription
RNA

Translation

John Donnie A. Ramos, Ph.D.

Dept. of Biological Sciences
College of Science
University of Santo Tomas

Protein

Modes of DNA Replication

Semiconservative Replication

Semiconservative Replication in Prokaryotes

Semiconservative Replication in Prokaryotes

Mathew Messelson and Franklin Stahl (1958)

15N

heavy isotope of N (contains 1 more

neutron) compared to 14N

15N

Expected results of the Messelson-Stahl experiment

has high sedimentation rate in cesium

chloride compared to 14N

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Semiconservative Replication in Eukaryotes

J. Herbert Taylor, Philip
Woods, and Walter Hughes
(1957)

Replication of E. coli Plasmid

Shown by John Cairns (1981) using
radioisotopes and radiography
Replication starts in a single OriC
origin of replication (245 bp)

Used root tip cells from Vicia

faba (broad bean)

Replication is bidirectional

Monitored replication using

3H-Thymidine to label DNA

Replication fork unwound DNA helix

Replicon replicated DNA

Used autoradiography to
determine the incorporation
of 3H-Thymidine

Ter region region of replication

termination

Arrested cells at metaphase

using colchicine

DNA Synthesis in Microorganisms

Chain Elongation
5 to 3 direction of DNA synthesis (requires 3 end of the DNA template)
Each step incorporates free 3 OH group for further elongation

DNA polymerase I (928

aa) catalyses the
synthesis of DNA in vitro
(A. Kornberg, 1957)
Requirements:
Deoxyribonucleoside
triphosphates, dNTPs
(dATP, dCTP, dGTP, dTTP)
DNA template
Primer

DNA replication using DNA polymerase is of high fidelity (highly

accurate)
With exonuclease activity (proofreading ability)

DNA Polymerases
All 3 types requires a primer
Complex proteins (100,000 Da)
Functions of DNA polymerases
in vivo
DNA Pol I proofreading;
removes primers and fills gaps
DNA Pol II - mainly involved in
DNA repair from external
damage

Replication in Prokaryotes
1.
2.
3.
4.
5.

Unwinding of DNA helix

Initiation of DNA synthesis
DNA synthesis proper (elongation)
Sealing gaps
Proofreading and error correction

DNA Pol III main enzyme

involved in DNA synthesis
a holoenzyme (>600,000 Da)
forms replisome when
attached to a replication fork.

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Unwinding of DNA Helix

Initiation of DNA Synthesis

Takes place in oriC (245 bp)

repeating 9mers and 13mers

Function of helicases (Dna A, B, C)

requires ATP hydrolysis to break
hydrogen bonds

Initiated by Dna A binds to 9mers

Binding of Dna B and Dna C to
unwound helix

Single-stranded binding proteins

(SSBPs) prevents reannealing of
replication bubble.

Synthesis of RNA primer 5 to 15 RNA bases complementary to

the DNA template

Catalysed by primase (an RNA polymerase)

Pimase does not require free 3 end to initiate synthesis (not unlike
DNA polymerase III)

DNA gyrase (a DNA topoisomerase)

Function of primase will be continued by DNA polymerase III.

relaxes the supercoiling of DNA

helix

DNA Synthesis (Elongation)

Function of DNA polymerase III
Requires free 3 end
Direction of elongation: 5 to 3
DNA synthesis is continuous in 3 to 5

DNA strand (leading strand) and

discontinuous in the 5 to 3 DNA strand
(lagging strand).

Okazaki fragments short DNA

fragments produced in the lagging

strand

Concurrent synthesis of leading and

lagging strands occur by using DNA pol

dimer and by a looping mechanism for
the lagging strand

Sealing of Gaps, Proofreading

and Error Correction
DNA polymerase I removes all RNA bases produced

by primase (creates gaps in the lagging strand) and

replaces it with DNA bases (U to T).

DNA ligase seals the gaps by forming

phosphodiester bonds

Exonuclease proofreading (identification of

mismatched bases) is a function of both DNA

polemerase I and III (both with 3-5 exonuclease
activity)

subunit of DNA polymerase III is involved in

proofreading.

Assures high fidelity of DNA replication

Mutations Affect Replication

Replication in Eukaryotes
Presence of multiple replication origin
(faster replication, guarantees
replication of a big genome) 25K
replicons in mammalian cells

Autonomously replicating sequences

(ARSs) origin of replication in yeasts

(11 bp)

Origin site is AT rich region

Helicase unwinds double stranded DNA

and removes histone proteins from DNA

Histones reassociates while DNA

synthesis occurs.

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Eukaryotic DNA Polymerases

Eukaryotic DNA Polymerases

Pol - initiates nuclear DNA synthesis

4 subunits (2 acts primase produces RNA primers)

Acts on both leading and lagging strands
2 other subunits continue elongation step (DNA synthesis)
Low processivity (short length of synthesized DNA prior to dissociation)

Pol - replaces Pol (called polymerase switching)

High processivity (during elongation)
With 3-5 exonuclease activity (proofreading)

Pol - nuclear DNA synthesis

Pol - DNA repair (the only eukaryotic DNA polymerase with single
subunit)

Pol - DNA repair

Pol - mitochondrial DNA synthesis (encoded by nuclear gene)
Eukaryotes has a high copy number of DNA polymerases (ex. Pol
may be up to 50K copies)

Eukaryotic DNA Replication

Telomerase
Enzyme that adds TTGGGG

Telomeres linear ends of

eukaryotic chromosomes

Problem with lagging

strand: no 3 needed by
DNA polymerase I (after
removal of RNA primers)

Possible result: chromosome

with shorter lagging strand
every replication step

repeats on the telomeres (first

identified in Tetrahymena)

Prevents shortening of
chromosomes

Forms a hairpin loop on

chromosome ends using G-G

bonds

Creates a free 3 on lagging

strand that can be used by

DNA polymerase I to replaced
the removed RNA primer

Telomerase is a

ribonucleoprotein and contains

RNA sequence (5 AACCCC 3serving as template) reverse
transcriptase

Cleavage of loop after DNA

synthesis

Homologous
Recombination
Exchange of genetic material
Directed by specific
enzymes:
Endonuclease introduces
single strand nicks

Gene Conversion
Exchange of genetic information between non-homologous
chromosomes (non-reciprocal genetic exchange)

Type of chromosome mutation (recombination)

First identified in Neurospora (by Mary Mitchell)
Can be repaired but forms recombined genetic material

Ligase seals loose ends

(nicks)

Rec A protein promotes the

exchange of reciprocal
single-stranded DNA
molecules and it enhances
hydrogen bond formation
during strand displacement

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Short Quiz

Short Quiz

Assume that the sequence of bases given below is present on one

Assume that the sequence of bases given below is present on one

nucleotide chain of a DNA duplex and that the chain has opened up
at a replication fork. Synthesis of an RNA primer occurs on this
template starting at the base that is underlined.

nucleotide chain of a DNA duplex and that the chain has opened up
at a replication fork. Synthesis of an RNA primer occurs on this
template starting at the base that is underlined.

3..GGC TAC CTG GAT TCA..5

3..GGC TAC CTG GAT TCA..5

a. If the RNA primer consists of 8 nucleotides, give the sequence

a. If the RNA primer consists of 8 nucleotides, give the sequence

of the primer (showing the 3 and 5 ends)

of the primer (showing the 3 and 5 ends)

b. In the intact RNA primer, which nucleotide has a free 3 OH

terminus?

c. Give the sequence of the complementary strand of the given

DNA.

d. How many hydrogen bonds are found in the given double

Given DNA strand

3..GGC TAC CTG GAT TCA..5

RNA Primer

5..AC CUA AGU..3

helical DNA strand?

e. Give the sequence of the primer that will be synthesized in the

complementary strand if the primer synthesis starts in the
complementary base of the underlined base above.

Answers

Short Quiz

b. In the intact RNA primer, which nucleotide has a free 3 OH

terminus?

Given DNA strand

3..GGC TAC CTG GAT TCA..5

RNA Primer

5..AC CUA AGU..3

c. Give the sequence of the complementary strand of the given

DNA.
Given DNA strand

3..GGC TAC CTG GAT TCA..5

Complementary Strand 5.CCG ATG GAC CTA AGT3

Uracil

Short Quiz

Short Quiz

d. How many hydrogen bonds are found in the given double

helical DNA strand?

Given DNA strand

3..GGC TAC CTG GAT TCA..5

Complementary Strand 5CCG ATG GAC CTA AGT3

Between C and G = 3 hydrogen bonds

= 8 x 3 = 24

Between A and T = 2 hydrogen bonds

= 7 x 2 = 14
______

Total hydrogen bonds

e. Give the sequence of the primer that will be synthesized in the

complementary strand if the primer synthesis starts in the
complementary base of the underlined base above.

38

Given DNA strand

3..GGC TAC CTG GAT TCA..5

Complementary Strand 5CCG ATG GAC CTA AGT3

RNA Primer

3..GGC UACCU5
or
5..UCC AUC GG..3