3/22/2016
1
3/22/2016
2
DNA is a long polymer made from repeating
units called nucleotides. The DNA chain is
22 to 26 Ångströms wide (2.2 to 2.6
nanometres), and one nucleotide unit is 3.3
Å (0.33 nm) long.
The backbone of the DNA strand is made
from alternating phosphate and 2-deoxyribose, a pentose. The pentoses are joined
together by phosphate groups that form
phosphodiester bonds between the third
and fifth carbon atoms of adjacent sugar
rings. These asymmetric bonds mean each
strand of DNA has a direction. In a double
helix the strands are antiparallel.
The asymmetric ends of DNA strands are
called the 5′ and 3′ ends, with the 5' end
having a terminal phosphate group and the
3' end a terminal hydroxyl group. One
major difference between DNA and RNA is
the sugar, with the 2-deoxyribose in DNA
being replaced by the alternative pentose
sugar ribose in RNA
3/22/2016
3
In DNA Replication an entire double-stranded DNA is copied to produce a
second, identical DNA double helix. In this process, many different proteins
which are clustered together in particular locations in the cell act concertedly.
The incoming DNA double helix is split into two single strands and each original
single strand becomes half of a new DNA double helix. Because each resulting
DNA double helix retains one strand of the original DNA, DNA replication is said
to be semi-conservative
These proteins are:
• Helicase – which unwinds the DNA double helix into two individual strands.
• Single-strand binding proteins (SSBs), which “coat” the single-stranded DNA
and prevent the DNA strands from reannealing to form double-stranded DNA.
• Primase is an RNA polymerase that synthesizes the short RNA primers
needed to start the strand replication process.
• DNA polymerase is an enzyme that strings nucleotides together to form a
DNA strand.
• The sliding clamp is an accessory protein that helps hold the DNA polymerase
onto the DNA strand during replication.
• RNAse H removes the RNA primers that previously began the DNA strand
synthesis.
• DNA ligase links short stretches of DNA together to create one long
continuous DNA strand.
3/22/2016
4
http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html
3/22/2016
5
Structure of E. coli helicase RuvA:
http://www.youtube.com/watch?v=JRGN35vM4Vw
http://www.youtube.com/watch#!v=AhTKDFxQneY&feature=related
3/22/2016
6
3/22/2016
7
Helicases are motor proteins that move directionally along a nucleic acid
phosphodiester backbone, separating two annealed nucleic acid strands (i.e.
DNA, RNA, or RNA-DNA hybrid) using energy derived from ATP hydrolysis.
Helicases adopt different structures and oligomerization states. Whereas
DnaB-like helicases unwind DNA as donut shaped hexamers, other enzymes
have been shown to be active as monomers or dimers. The common function of
helicases accounts for the fact that they display a certain degree of amino
acid sequence homology (similarities); they all possess common sequence motifs
located in the interior of their primary sequence. These are thought to be
specifically involved in ATP binding, ATP hydrolysis and translocation on the
nucleic acid substrate. The variable portion of the amino acid sequence is
related to the specific features of each helicase.
Based on the presence and the form of helicase motifs, helicases have been
separated in 4 superfamilies and 2 smaller families.
Some members of these families are indicated in the next slide, with the
organism from which they are extracted, and their function:
3/22/2016
8
Superfamily I: UvrD (E. coli, DNA repair), Rep (E. coli, DNA replication), PcrA
(Staphylococcus aureus, recombination), Dda (bacteriophage T4, replication
initiation), RecD (E. coli, recombinational repair), TraI (F-plasmid, conjugative DNA
transfer).
Superfamily II: RecQ (E. coli, DNA repair), eIF4A (Baker's Yeast, RNA
translation), WRN (human, DNA repair), NS3[4] (Hepatitis C virus, replication).
TRCF (Mfd) (E.coli, transcription-repair coupling).
Superfamily III: LTag (Simian Virus 40, replication), E1 (human papillomavirus,
replication), Rep (Adeno-Associated Virus, replication, viral integration, virion
packaging).
DnaB-like family: dnaB (E. coli, replication), gp41 (bacteriophage T4, DNA
replication),T7gp4 (bacteriophage T7, DNA replication).
Rho-like family: Rho (E. coli, transcription termination).
Note that these superfamilies do not subsume all possible helicases. For example
XPB and ERCC2 are helicases not included in any of the above families.
3/22/2016
9
Science 19 October 2007: Vol. 318. no. 5849, pp. 459
– 463 DOI: 10.1126/science.1147353
Structure of Hexameric DnaB Helicase and
Its Complex with a Domain of DnaG Primase
Scott Bailey, William K. Eliason, Thomas A. Steitz*
The complex between the DnaB helicase and
the DnaG primase unwinds duplex DNA at the
eubacterial replication fork and synthesizes the
Okazaki RNA primers. The crystal structures
of hexameric DnaB and its complex with the
helicase binding domain (HBD) of DnaG reveal
that within the hexamer the two domains of
DnaB pack with strikingly different symmetries
to form a distinct two-layered ring structure.
Each of three bound HBDs stabilizes the DnaB
hexamer in a conformation that may increase its
processivity.
Three
positive,
conserved
electrostatic patches on the N-terminal domain
of DnaB may also serve as a binding site for
DNA and thereby guide the DNA to a DnaG
active site.
3/22/2016
10
3/22/2016
11
STRAND SEPARATION
To begin the process of DNA replication, the two double helix strands are
unwound and separated from each other by the helicase enzyme. The point
where the DNA is separated into single strands, and where new DNA will be
synthesized, is known as the replication fork.
SSBs, quickly coat the newly exposed single strands and maintain the
separated strands during DNA replication. Without the SSBs, the
complementary DNA strands could easily snap back together. SSBs bind
loosely to the DNA, and are displaced when the polymerase enzymes begin
synthesizing the new DNA strands.
NEW STRAND SYNTHESIS
(i) The two single DNA strands can act as templates for the production of
two new, complementary DNA strands. Polymerase enzymes can
synthesize nucleic acid strands only in the 5’ to 3’ direction, hooking the
5’ phosphate group of an incoming nucleotide onto the 3’ hydroxyl group at
the end of the growing nucleic acid chain. Because the chain grows by
extension off the 3’ hydroxyl group, strand synthesis is said to proceed in
a 5’ to 3’ direction.
3/22/2016
12
3/22/2016
13
(ii) DNA polymerase can only extend a nucleic acid chain but cannot start one
from scratch. To give the DNA polymerase a place to start, an RNA
polymerase called Primase first copies a short stretch of the DNA strand.
This creates a complementary RNA segment, up to 60 nucleotides long that is
called a primer.
Now DNA polymerase can copy the DNA strand. The DNA polymerase starts
at the 3’ end of the RNA primer, and, using the original DNA strand as a guide,
begins to synthesize a new complementary DNA strand. Two polymerase
enzymes are required, one for each parental DNA strand. Due to the
antiparallel nature of the DNA strands, however, the polymerase enzymes on
the two strands start to move in opposite directions.
One polymerase can remain on its DNA template and copy the DNA in one
continuous strand. However, the other polymerase can only copy a short
stretch of DNA before it runs into the primer of the previously sequenced
fragment. It is therefore forced to repeatedly release the DNA strand and
slide further upstream to begin extension from another RNA primer. The
sliding clamp helps hold this DNA polymerase onto the DNA as the DNA moves
through the replication machinery. The sliding clamp makes the polymerase
processive.
3/22/2016
14
3/22/2016
15
Model for primase structure and function within the replisome. (Inset) Organization of the helicase and primase
components of the replisome as observed in the bacteriophage T7 primase-helicase polyprotein. Primase (purple)
directly abuts the helicase (gold). The lagging-strand DNA is thought to be threaded through the central channel.
(Left and right panels) Models for the orientation of DnaG with respect to DnaB. DNA is shown in blue with
synthesized RNA in red. Regions in gray denote the ZBD and DnaB-ID of full-length DnaG whose positions are
inferred from the location of the DnaG-RNAP NH2- and COOH-termini. (Left) The primase active site faces away
from the central hole of the helicase. ssDNA extruded from the helicase must loop back to reach the primase
active site. The direction by which the RNA:DNA hybrid is translocated and ssDNA is extruded are the same
(red and blue arrows, respectively). (Right) The DnaG active site faces toward the interior hole of the helicase.
Two DnaB protomers have been cut away to show the central hole, where ssDNA from DnaB is guided directly
into the DnaG catalytic center for transcription of RNA. The directions of RNA:DNA hybrid translocation and
incoming ssDNA are opposed (arrows). Such a model suggests that primer size preferences observed in vitro and
in vivo could arise, in part, from steric effects between the primase, helicase, and newly synthesized primer. The
directionality of nucleic acid binding to DnaG is indicated as discussed in the text; although a model where DnaGRNAP binds primer-template in a different configuration cannot be entirely excluded, existing observations agree
with the orientation shown.
3/22/2016
16
In molecular biology, processivity is a measure of the average number of
nucleotides added by a DNA polymerase enzyme per association/disassociation
with the template.
DNA polymerases associated with DNA replication tend to be highly processive,
while those associated with DNA repair tend to have low processivity. Because the
binding of the polymerase to the template is the rate-limiting step in DNA
synthesis, the overall rate of DNA replication during the synthesis (or “S”) phase
of the cell cycle is dependent on the processivity of the DNA polymerases
performing the replication. DNA clamp proteins are integral components of the
DNA replication machinery and serve to increase the processivity of their
associated polymerases.
Some polymerases add over 50,000 nucleotides to a growing DNA strand before
dissociating from the template strand, giving a replication rate of up to 1,000
nucleotides per second.
The continuously synthesized strand is known as the leading strand, while the
strand that is synthesized in short pieces is known as the lagging strand. The
short stretches of DNA that make up the lagging strand are known as Okazaki
fragments.
3/22/2016
17
THE LAGGING STRAND
Before the lagging-strand DNA exits the “replication factory”, its RNA
primers must be removed and the Okazaki fragments must be joined together
to create a continuous DNA strand.
The first step is the removal of the RNA primer. RNAse H, which recognizes
RNA-DNA hybrid helices, degrades the RNA by hydrolyzing its phosphodiester
bonds.
Next, the sequence gap created by RNAse H is then filled in by DNA
polymerase which extends the 3’ end of the neighboring Okazaki fragment.
Finally, the Okazaki fragments are joined together by DNA ligase that hooks
together the 3’ end of one fragment to the 5’ phosphate group of the
neighboring fragment in an ATP- or NAD+-dependent reaction.
3/22/2016
18
1. The process begins when the helicase enzyme unwinds the double helix to expose
two single DNA strands and create two replication forks. DNA replication takes place
simultaneously at each fork. The mechanism of replication is identical at each fork.
Remember that the proteins involved in replication are clustered together and
anchored in the cell. Thus, the replication proteins do not travel down the length of
the DNA. Instead, the DNA helix is fed through a stationary replication factory like
film is fed through a projector.
2. Single-strand binding proteins, or SSBs, coat the single DNA strands to prevent
them from snapping back together. SSBs are easily displaced by DNA polymerase.
3. The primase enzyme uses the original DNA sequence as a template to synthesize a
short RNA primer. Primers are necessary because DNA polymerase can only extend a
nucleotide chain, not start one.
4. DNA polymerase begins to synthesize a new DNA strand by extending an RNA
primer in the 5' to 3' direction. Each parental DNA strand is copied by one DNA
polymerase. Remember, both template strands move through the replication factory in
the same direction, and DNA polymerase can only synthesize DNA from the 5’ end to
the 3’ end. Due to these two factors, one of the DNA strands must be made
discontinuously in short pieces which are later joined together.
5. As replication proceeds, RNAse H recognizes RNA primers bound to the DNA
template and removes the primers by hydrolyzing the RNA.
6. DNA polymerase can then fill in the gap left by RNase H.
7. The DNA replication process is completed when the ligase enzyme joins the short
DNA pieces together into one continuous strand.
3/22/2016
19