MOLECULAR BIOLOGY

For Second Year

PART 1 MOLECULAR BIOLOGY

Transmission electron micrograph of human

DNA from a HeLa cell, illustrating a

replication fork characteristic of active DNA

replication.

DNA Replication

Lecture Contents

4.1 DNA Replication—Maintaining Genetic Information

4.2 Models of DNA Replication
Semiconservative, Conservative and Dispersive
4.3 Components Required for Prokaryotic DNA Replication

4.4 Continuous and Discontinuous Synthesis

Leading strand and Lagging strand
4.5 Summary
4.6 Study Questions

4.1 DNA Replication—Maintaining Genetic Information

DNA must be replicated so that the information it holds can be maintained and
passed to future cell generations, even as that information is accessed to guide the
manufacture of proteins.

4.2 Models of DNA Replication

All of the atoms of one strand Atoms of the two parental DNA
of the parent molecule are strands serve as a template for
transferred intact and two new progeny strands. The
without rearrangement to one two parental strands remain
strand of the progeny IDNA intact (without rearrangement)
molecule: the other strand is and remain together following
formed entirely of new atoms. replication, as do the two
progeny strands.

All of the atoms of each strand of the

parent molecule appear in the progeny
DNA, but they appear as large sections
scattered throughout the length of both
strands of the progeny DNA.
4.3 Components Required for Prokaryotic DNA Replication

For the replication of double-stranded DNA, the following components are

necessary:

1. Template DNA:
The parent sequence of nucleotides to be used as information in the synthesis of
the complementary strand.
Neither DNA polymerase I nor III is capable of synthesizing DNA de novo. The
enzymes will not place nucleotides in a random order.
A template is necessary, As the new DNA strand is being synthesized, each new
nucleotide to be added is matching to the nucleotide on the template strand based
on Chargaff base pairing.

2. Origin:
A specific sequence of nucleotides on the parent DNA that is recognized as the
initiation site of synthesis.

Eukaryotic
cells have
has a single origin site, multiple sites
referred to as (oriC) on each
chromosome,
. referred to as
origins of
DNA
3. Proteins:
DnaA,DnaB,DnaC Rep protein SSB
Needed to recognize help to uncoil the tightly Help to unwind
the origin packaged parent DNA the double helix

separate the strands

of the parent DNA

4. Nucleotides:
The synthesis of DNA requires four nucleotides in the deoxyribose triphosphate
form and four in the ribose triphosphate form.

Nucleotides need for DNA synthesis are

1. deoxyadenine 5'-triphusphate (dATP)

2. deoxyguanosine 5'-triphosphate (dGTP)

3. deoxycytidine 5' triphosphate (dCTP)

4. deoxythymine 5'-triphosphate (dTTP)

For the synrhesis of the primer RNA, the nucleotides needed are:
1. adenosine 5'-triphosphate (ATP)
2. guanosine 5'-triphosphate (GTP)
3. cytidine 5'-triphosphate (CTP)
4. uridine 5-triphusphate (UTP)

5. Enzymes: Many enzymes are needed for replication, but we will deal only with
the most important ones and in the order they are used:
 gyrases: Uncoil the DNA in preparation for the activity of the helicase(s)

helicases: Unwind the double helix.

Note 1
A site where DNA is locally opened, resembling a fork, is called a replication fork.

RNA polymerase and primase: Needed for the synthesis of RNA primers, a
requirement of DNA synthesis.

Note 2
DNA synthesis is initiated by synthesis, a short piece of RNA, called primer
RNA because it primes DNA synthesis, is needed before DNA chain formation
can begin. The primer varies in length in different cell types, but on the order of
2-10 nucleotides.

Note 3
These enzymes initiate synthesis and elongate chain only in 5’ to 3’ direction.

DNA polymerase III: The actual replicating enzyme that synthesizes DNA
it takes its instructions from the parental- template DNA strand. Using the RNA
primer as an anchor and the triphosphate deoxyribonucleotides (dATT dGTP,
dCTP, and dTTT), DNA polymerase adds deoxyribonucleotides to the primer,
thereby elongating the chain.
Note 4
As the enzyme moves along the parent strand, it "reads" each parent nucleotide
and add new nucleotide to the growing DNA chain. Synthesis is always in the
5' to 3' direction.

DNA polymerase I: Removes the RNA primers and replaces them with DNA. Is
also the "proofreading" enzyme
DNA polymerase I is the second of the two DNA synthesizing enzymes.
polymerase I is essential for both the synthesis and the repair of DNA.
DNA polymerase I has, in fact, three enzyme activities:
I. DNA polymerization
2. depolymerization in a 5' to 3' direction
3. depolymerization in a 3' to 5' direction, the proofreading activity

The last two activities are referred to as exonuclease activity, which means that the
enzyme is able to remove nucleotides, one at a time, from either the 5' or the 3' end
of nucleic acid chains.

DNA ligase: Joins the Okazaki fragments by way of phosphodiester bond.

function:
The DNA ligases are responsible for connecting DNA segments during
replication, repair, and recombination.

All ligases join a 5'- phosphoryl group and a 3'-phosphoryl group on adjacent
fragments, thereby sealing the nick.

4.4 Continuous and Discontinuous Synthesis

DNA synthesis process has two way for synthesis newly strands

Continuous Synthesis Discontinuous Synthesis

one strand is elongated the other new strand is produced by

by the continuous repeated synthesis of primer RNAs
additions of nucleotides and short lengths of DNA (Okazaki
to the 3' end. fragments), which must eventually
be joined by DNA ligase.
 this newly synthesized DNA this newly synthesized DNA
is called the leading strand is called the lagging strand

Note 5
Because DNA synthesis is continuous on one strand and discontinuous on the
other, the term semidiscontinuous synthesis is sometimes used to describe the
overall process.

Note 6
The two strands of DNA are antiparallel; they run in opposite directions Also, keep
in mind that DNA synthesis is always in the 5' to 3' direction.

opposite polarity of synthesis

Semidiscontinuous repilcation
4.5 Summary

During replication, the DNA unwinds locally at several sites. Replication forks
form as hydrogen bonds break between base pairs. Primase builds short RNA
primers, which DNA sequences eventually replace. Next, DNA polymerase fills in
DNA bases, and ligase seals the sugar-phosphate backbone.
Replication proceeds in a 5 ′ to 3 ′ direction, so the process must be discontinuous
in short stretches on one strand.

4.6 Study Questions

1. Briefly explain the semiconservative model of DNA.

2. Describe the role of each of the following in prokaryotic DNA replication:
triphosphate nucleotides, template DNA, primase, RNA primer, DNA polymerases I
and III, and DNA ligase.
3. Describe how leading and lagging strand synthesis are similar and different.
4. What are Okazaki fragments?
5. What is the direction of DNA synthesis?