semiconservative replication
-each of the original nucleotide strands remains intact (conserved), despite no longer being combined in the same molecule; the original DNA molecule is half (semi) conserved during replication
-only one sequence of bases can be specified by each DNA stra
What did the complementary nature of the two nucleotide strands in a DNA molecule suggest about replication?
That during replication each strand can serve as a template for the synthesis of a new strand
conservative replication
entire double-stranded DNA molecule serves as a template for a whole new molecule of DNA, and the original DNA molecule is fully conserved during replication
dispersive replication
both nucleotide strands break down (disperse) into fragments, which serve as templates for the synthesis of new DNA fragments, and then somehow reassemble into two complete DNA molecules, each resulting DNA molecule is interspersed with fragments of old a
How was the mode of DNA replication discovered?
By using heavy and light nitrogen isotopes, and centrifugation
How many bands of DNA would be expected in Meselson and Stahl's experiment after two rounds of conservative replication?
two
replicon
individual unit of replication, containing a replication origin
Where does replication start in a replicon and where does it stop?
starts at the origin of replication, continues until the entire replicon has been replicated
Theta replication
takes place in circular DNA, unwinding at the replication origin occurs, creating single stranded nucleotide strands (template), unwinding can occur at one or both ends of the replication bubble (uni or bi directional, one or two replication forks), DNA r
replication bubble
loop generated by the unwinding of the double helix
replication fork
point of unwinding in a replication bubble where the two single nucleotide strands separate from the double stranded DNA helix
bidirectional replication
occurs if there are two replication forks, one at each end of the replication bubble, allow for replication to proceed outward in both directions
rolling circle replication
takes place in some viruses, and in the F factor; initiated by a break in one of the nucleotide strands that creates a 3'-OH group and a 5'-phosphate group; new nucleotides are added to the 3' end of the broken strand, the inner unbroken strand remains as
Linear Eukaryotic Replication
larger chromosomes, requires multiple origins of replication, at each replication origin DNA unwinds and produces a replication bubble, replication takes place bidirectionally, replication forks of adjacent replicons run into each other, and the replicons
Which type of replication requires a break in the nucleotide strand to get started?
rolling circle replication
Theta vs. Rolling Circle vs. Linear Eukaryotic Replication:
DNA Template, Breakage of Nucleotide Strand, Number of Replicons, Unidirectional or Bidirectional, Products
Theta: circular, no breakage, one replicon, uni or bidirectional, two circular molecules
Rolling Circle: circular, breakage, one replicon, unidirectional, one circular molecule, and one (or many) linear molecule that may circularize
Linear Eukaryotic: lin
three requirements of replication:
1.) a template consisting of single-stranded DNA (or RNA)
2.) raw materials (substrates) to be assembled into a new nucleotide strain (dNTPs)
3.) enzymes and other proteins that "read" the template and assemble the substrates into a DNA molecule, primer,
dNTP
deoxyribonucleoside triphosphate, raw material from which new DNA molecules are synthesized, consists of a sugar and base (nucleoside), attached to three phosphate groups
Why does DNA unwind during replication?
to expose the bases that act as a template for the assembly of new polynucleotide strands, this leads to the semiconservative nature of DNA
How are nucleotides added to the growing strand in DNA synthesis?
dNTP's are added to the 3'-OH end of the growing strand, the 3' of the last nucleotide on the strand attacks the 5'-phosphate group of the incoming dNTP, two phosphate groups are cleaved from the incoming dNTP and a phosphodiester bond is created between
Does DNA synthesis occur spontaneously?
No, it requires several enzymes and proteins
DNA polymerases
enzymes that synthesize DNA, can only add nucleotides to the 3' end of growing strand (never the 5' end)
What is the direction of DNA elongation of the new strand?
5' to 3'
What does the antiparallel nature of DNA tell us about the direction of synthesis on the two strands?
DNA synthesis takes place in opposite directions on the two DNA template strands
continuous replication
occurs on the leading strand, synthesis occurs in the direction of unwinding
leading strand
undergoes continuous replication in the 5' to 3' direction, the template strand is 3' to 5'
discontinuous replication
synthesis occurs in the opposite direction of unwinding on the lagging strand
lagging strand
discontinuous replication occurs, has Okazaki fragments
Okazaki fragments
short lengths of DNA produced by discontinuous replication of the lagging strand, later linked together to create a continuous DNA molecule
Discontinuous replication is a result of which property of DNA?
antiparallel nucleotide strands
Which lagging strand fragments of DNA have been synthesized most recently?
Those nearest the replication fork
On which strand does DNA synthesis occur in the same direction as unwinding?
On the template leading strand
Is there a replication bubble in all DNA synthesis?
No, the rolling circle model does not involve the formation of a replication bubble
Does all DNA synthesis involve a leading and a lagging strand?
No, the rolling circle model has only continuous replication
DnaA
initiator protein in bacterial replication, binds to oriC (origin of replication sequence in E.coli). It causes a short section of DNA to unwind, which allows helices and SSBs to attach to the polynucleotide strand
What are the four stages of replication?
1.) initiation
2.) unwinding
3.) elongation
4.) termination
DNA helicase
part of unwinding, binds to unwind, single stranded DNA, breaks the hydrogen bonds between the bases of the two nucleotide strands of double stranded DNA, can NOT initiate unwinding, requires DnaA. Binds to the lagging strand template at each replication
Which strand of DNA in the replication bubble does DNA helcase bind to?
the lagging strand template at each replication fork, moving in 5' to 3' direction, moving the replication fork
single strains binding proteins
SSBs, attach tightly to exposed single stranded DNA AFTER it is unwound by helicase, protect single strand nucleotide chains and prevent formation of secondary structures (hairpins) which would interfere with replication, unspecific binding, form tetramer
DNA gyrase
a topoisomerase, enzyme involved in unwinding process, controls the supercoiling of DNA by reducing the torsional strain that builds up ahead of the replication fork as a result of unwinding. It makes a double stranded break in one segment of the DNA heli
What proteins/ enzymes take place in bacterial initiation?
DnaA
What proteins/ enzymes take place in bacterial unwinding?
DNA helicase, SSBs, DNA gyrase
Place the following components in order in which they are first used in the course of replication: helicase, single-strand-binding protein, DNA gyrase, DnaA
DnaA, helicase, SSBs, DNA gyrase
What proteins/ enzymes take place in bacterial elongation?
DNA primase, primer, DNA polymerase III, DNA polymerase I, DNA polymerase II, IV, and V, DNA ligase
primase
enzyme that synthesizes short stretches of RNA nucleotides, primers, to get DNA replication started, it is an RNA polymerase so it does not require a 3'-OH to add nucleotides
primers
short stretches of RNA nucleotides that provide 3'-OH for DNA polymerase to attach to and add nucleotides to
How many primers are on the leading strand?
Only one RNA primer at the origin of replication
Where does primase exist in replication?
Primase forms a complex with helices at the replication fork and moves along the template of the lagging strand
Primers are synthesized where on the lagging strand?
at the beginning of every Okazaki fragment
DNA polymerase III
E.coli polymerase, catalyzes DNA polymerization, large multiprotein complex, adds new nucleotides to the 3' end of a growing DNA molecule. It has 5' to 3' polymerase activity, and 3' to 5' exonuclease activity (to correct errors) has a high processivity (
3' to 5' exonulcease activity
removes nucleotides in the 3' to 5' direction
What ensures the high processivity of DNA polymerase III?
the beta subunit, which is one of the polypeptides of the enzyme, it serves as a clamp for the polymerase enzyme, encircling the DNA and keeping the DNA polymerase attached to the template strand during replication
DNA polymerase I
E.coli polymerase, catalyzes DNA polymerization; 5' to 3' polymerase, and 3' to 5' exonuclease activity, can synthesize DNA and correct for errors, and 5' to 3' exonuclease activity (or RNase H), can remove the primers laid down by primase and replace the
Functions of all polymerases of e.coli:
1.) synthesis of any sequence specified by template strand
2.) synth in 5' to 3' direction by adding nucleotides to 3' OH group
3.) use of dNTPs to synthesize new DNA
4.) require primer to initiate synthesis
5.) catalyze the formation of a phosphodiester
Characteristics of DNA Polymerases of E. coli:
DNA polymerase, 5' to 3' polymerization, 3' to 5' exonuclease, 5' to 3' exonuclease, function
I: yes, yes, yes, removes and replaces primers
II: yes, yes, no, DNA repair...
III: yes, yes, no, elongates DNA
IV: yes, no, no, DNA repair
V: yes, no, no, DNA repair...
DNA ligase
seals the nick left in the sugar phosphate backbone after the replacement of the RNA primers by DNA polymerase I, by catalyzing the formation of a phosphodiester bond
Which bacterial enzyme removes the primers?
DNA polymerase I
What proteins/ enzymes take place in bacterial termination?
Tus, protein that binds to certain sequences blocking the movement of helicase, stalling the replication fork and preventing further replication
What causes termination?
- when two replication forks meet
- specific termination sequences block further replication
proofreading
corrects errors that arise in nucleotide selection during DNA synthesis, occurs because of misplaced 3'OH group for next nucleotide attachment
mismatch repair
corrects errors after replication is complete, requires the ability to distinguish between old and new strands, can do this because only the old DNA strand is methylated
What causes the high fidelity of DNA replication?
proofreading, nucleotide selection, and mismatch repair
Which mechanism requires the ability to distinguish between newly synthesized and template strand of DNA?
Mismatch repair
autonomously replicating sequences
(ARSs) enable any DNA to which they are attached to replicated, origin of replication
origin recognition complex
(ORC), binds to origins of replication and unwinds the DNA in this region
In comparison with prokaryotes what are some differences in the genome structure of eukaryotic cells that affect how replication takes place?
The size of the eukaryotic genomes, the linear structure of eukaryotic chromosomes and the association of DNA with histone proteins
replication licensing factor
attaches to an origin, licensing the origin in approval for replication, occurs early in the cell cycle, removed once the replication fork moves aways from it
MCM
minichromosome maintenance, the eukaryotic licensing factor, contains a DNA helicase
DNA polymerase alpha
eukaryotic DNA polymerase, contains primase activity and initiates nuclear DNA synthesis by synthesizing an RNA primer followed by a short string of DNA nucleotides
What are the three eukaryotic DNA polymerases that carry out most of nuclear DNA synthesis during replication?
DNA polymerase alpha
DNA polymerase delta
DNA polymerase epsilon
DNA polymerase delta
eukaryotic DNA polymerase, completes replication on the lagging strand of DNA
DNA polymerase epsilon
eukaryotic DNA polymerase, complete replication on the leading strand
DNA polymerases in eukaryotic cells of alpha, delta, epsilon: 5' to 3' polymerase activity, 3' to 5' exonuclease activity, cellular function
alpha:yes, no, initiation of nuclear DNA synthesis and DNA repair, has primase activity
delta: yes, yes, lagging strand synthesis of nuclear DNA, DNA repair, and translesion DNS synthesis
epsilon:yes, yes, leading strand synthesis
translesion DNA polymerases
lower fidelity, are able to accommodate and copy templates with abnormal bases, distorted structures, and bulky lesions, its errors can lead to mutations if not caught
Some of the eukaryotic DNA polymerases have a tendency to make errors in replication. Why would a cell use an error prone DNA polymerase instead of one that is more accurate?
Because error prone DNA polymerases can bypass bulky lesions in the DNA helix that stall accurate high speed DNA polymerases
What is semiconservative replication?
original two strands of the double helix serve as templates for new strands of DNA, when replication is complete, two double stranded DNA molecules will be present, each will consist of one original template strand and one newly synthesized strand that is
How did Meselson and Stahl demonstrate that replication in E. Coli takes place in a semiconservative manner?
Grew E. coli cells in a medium containing the heavy isotope of nitrogen (N15) for several generations. E. coli cells are then switched to a medium containing the common form of nitrogen (N14) and allowed to proceed through a few cycles of cellular generat
What are three major requirements of replication?
1.) a single stranded DNA template
2.) nucleotide substrates for synthesis of the new polynucleotide strand
3.) enzymes and other proteins associated with replication to assemble the nucleotide substrates into a new DNA molecule
What substrates for DNA synthesis are used in the DNA synthesis reaction?
four types of deoxyribonucleoside triphosphates,
deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytosine triphosphate, and deoxythymidine triphosphate
List the different proteins and enzymes taking part in bacterial replication. Give the function of each in the replication process.
DnaA: binds to the replication origin and unwinds short regions of DNA
DNA polymerase III: primary replication polymerase, elongates new nucleotide strand from the primer
DNA polymerase I: removes RNA nucleotides of the primers and replaces them with DNA
What similarities and differences exist in the enzymatic activities of DNA polymerases I, II, and III? What is the function of each type of DNA polymerase in bacterial cells?
Each has a 5' to 3' polymerase activity, differ in their exonuclease activity, DNA polymerase II and III have only a 3' to 5' exonuclease activity
1.) poly I: carries out proofreading, and removes and replaces the RNA primers used to initiate DNA synthesi
Why is primase required fore replication?
Is a DNA-dependent RNA polymerase. It synthesizes short RNA molecules (primers) with a free 3'-OH that dNTPs can be attached to. DNA polymerases require a free 3'-OH to which they add nucleotides and therefore they cannot initiate replication. Primase doe
What three mechanisms ensure the accuracy of replication in bacteria?
1.) Highly accurate nucleotide selection by the DNA polymerases when pairing bases
2.) proofreading function of DNA polymerases which removes incorrectly inserted bases
3.) mismatch repair apparatus that repair mistakes after replication is complete
In what ways is eukaryotic replication similar to bacterial replication, and in what ways is it different?
Same: semiconservative replication, replication origins serve as starting points for replication, short segments of RNA (primers) provide 3' OH for DNA polymerases to begin synthesis of the new strand, synthesis occurs in a 5' to 3' direction, template st
What is the end of chromosome problem for replication? Why, in the absence of telomerase, do the ends of chromosomes get progressively shorter each time the DNA is replicated?
For DNA polymerases to work, they need a 3'-OH group to add a nucleotide to. At the ends of chromosomes when the RNA primers is removed there is no adjacent 3' OH group to which to add a nucleotide, thus no nucleotides are added leaving a gap at the end o
What happens to histones in replication?
They are removed and redistributed on the new DNA, which will contain nucleosomes contained of new and old histones.
end of replication problem:
occurs in linear chromosomes during replication, elongation of DNA in adjacent replicons provides a 3'-OH group, at the very end of a linear chromosome there is no adjacent OH group, once the primer at the end of the chromosome has been removed, it can NO
telomerase
an enzyme with both a protein and an RNA component (ribonucleoprotein) that is complementary to the overhanging piece of chromosome, this sequence pairs with the overhanging 3' end of the DNA and provides a template for the synthesis of additional DNA cop
In which types of cells does chromosome shortening NOT take place?
single celled organisms, germ cells, early embryonic cells due to telomerase
What would be the result if an organisms telomerase were mutated and nonfunctional?
Chromosomes would shorten with each new generation
genome size=
number of ori x length of replicon in bp
exonuclease
cleaves phosphodiester bonds at end
endonuclease
cleaves phosphodiester bonds from with in DNA molecule
What does it mean to be a DNA dependent DNA polymerase vs. an RNA dependent DNA polymerase?
DNA dependent uses DNA as a template, RNA dependent uses RNA as the template to generate DNA (i.e. telomerase)
Which polymerase exists as a dimer and what does this mean?
DNA polymerase III, or E.coli, is the trombone model, 2 DNA pol III are connected on the leading and lagging strand, so synthesis occurs in the same direction. In order for this to occur the lagging strand loops around so synthesis can occur in the 5' to
processivity vs. dispersive
Dispersive polymerases add a few units then fall off (lower processivity) more errors and slower
beta-clamp in e.coli holds DNA pol III on to result in high processivity
misincorporation
wrong base inserted in DNA synthesis