DNA replication- Chapter 7
What is ARS in yeast:
Bascially ORI for yeasts.
Summarize Topoisomerases
act as swivels to allow DNA to rotate during replication
How does DNA polymearse III intiate DNA synthesis of the lagging strand?
binding to the 3' end of an RNA primer, it synthesizes a DNA copy in a 5' to 3' direction until it reaches another okazaki fragment where it dissociates BUT REMAINS ATTACHED TO CLAMP-LOADING PROTEN Clamp-laoding protein places DNA polymerase to the next primer
How are sliding-clamp proteins (PCNA) loaded onto DNAat the junction between the primer and the template?
by clamp-loading proteins (RFC in eukaryotes)
In eukaryotic cells, what synthesizes the lagging strrand?
complex of primase with polymerase a (see figure 7.6= very helpful)
How does RFC load the PCNA?
using energy derived from ATP hydrolysis to open the sliding clamps of PCNA. RFC then released PCNA, which forms a ring around template DNA (hugging it). Summary: RFC puts PCNA to DNA PCNA hangs to DNA RFC released
How many replication fork per ORI?
• At a given ori, once unwind, you generate two replication forks
Describe the ORI of e.coli:
• Replication initiates at a unique site on the E. coli chromosome, designated the origin (ori). The first event is the bind-ing of an initiator protein to ori DNA, which leads to partial unwinding of the tem-plate. The DNA continues to unwind by the actions of helicase and single-stranded DNA-binding proteins, and RNA primers are synthesized by primase. The two replica-tion forks formed at the origin then move in opposite directions along the circular DNA molecule.
Give an example of proofreading
G is incorporated in place of A as a result of mispairing with T on the template strand. Because it is mispaired, the 3′ terminal G is not hydrogen-bonded to the template strand. This mismatch at the 3′ terminus of the growing chain is recognized and excised by the 3′ to 5′ exonuclease activity of DNA polymerase, which requires a primer hydrogen bonded to the tem-plate strand in order to continue synthesis. Following excision of the mismatched G, DNA synthesis can proceed with incorporation of the correct base (A) see figure 7.11
Is RNA primase attached to the RFC clamp loading protein?
No, but helicase is binded with RNA primase so it is indirectly
What is the RFC clamp loading protein binded to?
Polymerase enzymes helicase
4. What is the function of 3′ to 5′ exonuclease activity in DNA polymer-ases? What would be the consequence of mutating this activity of DNA polymerase III on the fidelity of replication of E. coli DNA?
3′ to 5′ exonuclease activity is required for the ex-cision of mismatched bases in newly synthesized DNA during proofreading. A mutant E. coli with a DNA polymerase III lacking this activity would have a high frequency of mutations each time the DNA is replicated
DNA strands are antiparallel, what is the overall direction for both strands in replication?
5' to 3' moving in the same direction dNA polyermase is not moving in opposite direction
In bacteria, what removed RNA primers? How does it do it?
DNA polymerase I acts as an exonuclease that can hydrolyze DNA (or RNA) in either the 3' to the 5' Or the 5' to 3' direction. The action of DNA polymerase I as a 5′ to 3′ exonuclease removes ribonucleotides from the 5′ ends of Okazaki fragments, allowing them to be replaced with deoxyribonucleotides to yield fragments consisting entirely of DNA
1What is the role of DNA polymerase I in E. coli? Why do mammalian cells not need an analogous enzyme?
DNA polymerase I degrades RNA primers in E. coli. In mammalian cells, this is performed by RNase H.
In bacteria cells, what synthesizes the leading strrand?
DNA polymerase III note: in bacteria cells, DNA polymerase III synthesizes both LEADING AND LAGGING (see figure 7.6= very helpful) In E. coli, polymerase III is the major replicative polymerase, functioning in the synthesis both of the leading strand of DNA and of Okazaki fragments by the extension of RNA primers.
what enzyme can strart from scratch, does not need a primer?
RNA polyemerase
So, what is the main function of DNA polymerase I?
Removal of RNA primers via catalyitic exonucleases addition of DNA nuceltoieds (Filling in, or maybe this is DNA polymerase III)
What follows helicases?
The unwound DNA strands are then stabilized by single-stranded DNA-binding proteins so that they can serve as templates for new DNA synthesis. stabilize the unwound template DNA, keeping it in an extended single-stranded state so that it can be copied by the DNA polymerase
How is ORI recognized in eukaryotes?
Using ORC For yeast: ARS has a core sequence has been found to be the binding site of a protein complex (called the origin recognition complex, or ORC) that is required for initiation of DNA replication.
What are the function of topoisomerases?
WORKS AHEAD OF THE REPLICATION FORK catalyze the reversble breakage and rejoining of DNA strands. The transient breaks introduced by these enzymes serve as swivels that allow the two strands of DNA to rotate freely around each other The breaks introduced by topoisomerases serve as "swivels" that al-low the two strands of template DNA to rotate freely around each other so that replication can proceed without twisting the DNA ahead of the fork
Do eukaryotic cells require topoisomerases, given that eukaryotic chromosomes are linear rather than circular
Yes otherwise the complete chromosomes would have to rotate continously during DNA replciation/synthesis
Is helicase ahead of the replication fork?
Yes. Helicases unwind the two strands of parental DNA ahead of the replication fork.
5. How would you test a sequence of DNA in a yeast cell to determine whether it contains an origin of replication?
You would insert the DNA sequence into a plas-mid that lacks an origin of replication and deter-mine whether the recombinant plasmid is able to transform mutant yeast that require a plasmid gene for their growth. Only plasmids with a func-tional origin will yield a high frequency of trans-formed yeast colonies.
If DNA polymerase III disscoiates from DNA once it reaches the end of a okazaki fragment, how does it reasscoiaate to another okazaki fragment
• Because it is bound to the clamp loader, it can then be efficiently reloaded onto a new sliding clamp at the primer-template junction to initiate synthesis of another Okazaki fragment.
How does replication begin in eukaryotes?
• Replciaiton proceeds in both oppssite directions- make it far more effeicient- happens in both eukaryotes and prokaryotes
How was the first ORI discovered?
• The first origin to be defined was that of E. coli, in which genetic analysis indicated that replication always begins at a unique site on the bacterial chromosome. The E. coli origin has since been studied in detail and found to consist of 245 base pairs of DNA, elements of which serve as binding sites for proteins required to initiate DNA replication (
How is DNA replication iniiated in E.coli bacterial cells?
• The key step is the binding of an initiator protein to specific DNA sequences within the origin. o The initiator protein begins to unwind the origin DNA and recruits the other proteins involved in DNA synthesis. o Helicase and single-stranded DNA-binding proteins then act to continue unwinding and exposing the template DNA, and primase initiates the synthesis of leading strands. Two replication forks are formed and move in opposite directions along the circular E. coli chromosome.
How does the lagging strand maintain the overall direction of replication?
• The lagging-strand template is folded at the replication fork so that the polymerase subunit engaged in lagging-strand synthesis moves in the same overall direction as the other subunit, which is synthesizing the leading strand
What is proofreading in terms of DNA polymerase III
• The other major mechanism responsible for the accuracy of DNA replication is the proofreading activity of DNA polymerase. The replicative DNA polymerases (eukaryotic polymerases δ and ε and E. coli polymerase III) have an exonuclease activity that can hydrolyze DNA in the 3′ to 5′ direction. o This 3′ to 5′ exonuclease operates in the reverse direction of DNA synthesis, and participates in proofreading newly synthesized DNA (Figure 7.11).
In eukaryotic cells, how are RNA primers removed?
combined action of RNase H, an enzyme that degrades the RNA strand of RNA-DNA hybrids, and 5′ to 3′ exonucleases. BUT, it does not fill the resulting gaps. This is done by DNA polymearse delta (RNASE does not fill the gaps)
What happens to DNA polymerase III when it reaches the end of an okazaki fragment?
it dissociates from DNA.
How does the origin recognition complex (ORC) work?
it is a multisubunit complex that specifically recogninzes a sequences within the ARS/ORI sequence The ORC proteins function to recruit other proteins (including the MCM DNA helicase) to the origin, leading to the initiation of replication.
When PCNA is released from RFC, what happens to RFC
it stays around, waiting to clamp again
What happens to nucleosomes during DNA replication?
nucleosomes are also disrupted during DNA replication. The nucleosomes of the parental chromatin are divided between the two daughter strands of DNA, and new histones are then added to reassemble nucleosomes by additional proteins (chromatin assembly factors) that travel along with the replication fork. o histones are simply deassembled and reassembled
How is proofreading important in increasing fidelity of DNA replication?
o Proof-reading is effective because DNA polymerase requires a primer and is not able to initiate synthesis de novo. Primers that are hydrogen-bonded to the template are preferentially used, so when an incorrect base is incorporated, it is likely to be removed by the 3′ to 5′ exonuclease activity rather than being used to continue synthesis. o The 3′ to 5′ exonucleases of the replicative DNA polymerases selectively excise mismatched bases that have been incorpo-rated at the end of a growing DNA chain
Where are the adughter strand ssynthesized?
replication fork; representing region of active DNA snythesis
• Since DNA polymerase requires a primer and cannot initiate synthesis de novo, how is the synthesis of Okazaki fragments initiated?
short fragments of RNA serve as primers for DNA replication (Figure 7.4). The synthesis of RNA can initiate de novo, and an enzyme called primase synthesizes short fragments of RNA (e.g., three to ten nucleotides long) complementary to the lagging strand template at the replication fork. Okazaki fragments are then synthesized via extension of these RNA primers by DNA polymerase. So basically, the RNA primer services as an initiation start for the lagging strand where DNA polymerase can add DNA nucletoides as fragments. See figure 7.4, very important
Where does replication occur for both prokaryotic and eukaryotic dNA
sites called origin of replications (ORI); binding site for proteins to intiate replication
What maintains the association of DNA polymerease with tempalte DNA?
sliding-clamp proteins (PCNA in eukaryotes), these load the DNA polymerase onto the primer and maintain its stable association with the template. (DONUT LOOKING) The ring formed by PCNA maintains association of polymerase with its template as replication proceeds, allowing the uninterrupted synthesis of thousands of nucleotides DNA.
Are ORC conserved?
the role of ORC proteins as initia-tors of replication is conserved in all eukaryotes, from yeasts to mammals.
How many ORI are in E.Coli:
there is only one, because DNA polymearse super fast.
In eukaryotic cells, what synthesizes the leading strrand?
DNA polymerase epsilon (see figure 7.6= very helpful)
What are two main fundemenatal properrties all DNA polymerases have?
1. All polymerase synthesize DNA only in 5' to 3' direction by adding dNTP to the 3'OH of a growing chain. 2. DNA polymearses can add new dNTPS only to primer strand that is h-bonded to the template (they are not able to intiate DNA synthesis de novo by catalyzing the polymerization of free DNTPS. (unlike RNA polymerase)
What happens to the two strands of template DNA as they begin to unwind, in the absence of toposiomerases?
As the two strands of template DNA unwind, the DNA ahead of the replication fork is forced to rotate in the opposite direction, causing circular molecules to become twisted around themselves. • As the strands of parental DNA unwind, the DNA ahead of the replication fork is forced to rotate. o Unchecked, this rotation would cause circular DNA molecules to become twisted around themselves, eventually blocking replication
3. You are interested in studying the role of RNA polymerase in DNA replica-tion, so you treat cells with an inhibitor of RNA polymerase and determine its effects on DNA synthesis at the replication fork. How do you expect it to affect synthesis of the leading and lagging strands of DNA?
An inhibitor of RNA polymerase would block synthesis of the lagging strand by preventing syn-thesis of RNA primers. Synthesis of the leading strand would not be affected REMEMBER, PRIMASE IS AN RNA POLYMERASE
Describe the identification of origins of replication in yeast
Both plasmids I and II contain a selectable marker gene (LEU2) that allows transformed cells to grow on medium lacking leucine. Only plasmid II, however, contains an origin of replication (ARS). The transformation of yeasts with plasmid I yields only rare transformants in which the plasmid has integrated into chromosomal DNA. Plasmid II, however, is able to replicate without integration into a yeast chromosome (autonomous replication), so many more transformants result from its introduction into yeast cells. Summary: • ARS: promotes replication • Two different experimental groups • Transformed yeast in plasmid into a gene that make amino acid leucine • Grow on growth media that lack leucine • yeast cell that will only survive, ones that produce leucine • they need to replicate DNA to make a lot of copies of the gene to make leucine to survive • Rare event if plasmid integrates to yeast genome, so yeast that dont have ARS die off. • ARS is ORI for yeast • Identified ARS by survival of transformed yeast
What increases the fidelity of DNA replication
Complementary base pairing (h-bonding) base selection (conformation change) proofreading DNA mismatch repair
In Euakryotic cells, what fills the resulting gaps after the removal of RNA primers using RNASe H?
DNA polymerase delta (see figure 7.6= very helpful)
How were the ORI of eukaryotic chromosmomes first studied?
In yeasts. in which ORI were identified as sequences that can support the replication of plasmids in transformed cells (Figure 7.14). autonomously replicating sequences, or ARSs) have been isolated. Their role as origins of replication has been verified by direct biochemical analysis, not only in plasmids but also in yeast chromosomal DNA.
6. How would an inhibitor of telomerase affect replication of eukaryotic cells? Would it affect bacteria?
Inhibition of telomerase would block synthesis of the ends of linear chromosomes, which would be lost as eukaryotic cells replicate. Bacteria would be unaffected since they have circular genome.
How many ORI do eukaryotic cells have?
Many DNA synthesis is initiated at multiple sites, from which it then proceeds in both directions along the chromosome Replication origins in eukaryotic chromosomes Replication initiates at multiple origins (ori) in eukaryotic chromosomes, each of which produces two replication forks.
How does DNA polymerase increases the fidelty of replciation in terms of base selection?
The polymerase does not simply catalyze incorporation of whatever nucleotide is hydrogen bonded to the template strand. o Instead, it actively discriminates against incorporation of a mismatched base by adapt-ing to the conformation of a correct base pair (CHANGING SHAPE).
How is ORC in eukaryotes similar to intiator in bacteria>
The mechanism of initiation of DNA replication of eukaryotes appears similar to that inbacteria and eukaryotic viruses; that is, an initiator protein binds to specific DNA sequences that define an origin of replication.
• Since the two strands of double-helical DNA run in opposite (antiparallel) directions, continuous synthesis of two new strands at the replication fork would require that one strand be synthesized in the 5′ to 3′ direction while the other was synthesized in the opposite (3′ to 5′) direction. • But DNA polymerase catalyzes the polymerization of dNTPs only in the 5′ to 3′ direction. How, then, can the other progeny strand of DNA be synthesized?
• This enigma was resolved by experiments showing that only one strand oF DNA synthesis is initiated with RNA primers that are later replaced with DNA (LAGGING STRAND). • DNA is synthesized in a continuous manner in the direction of overall DNA replication; the other is formed from short (1-3 kb), discontinuous pieces of DNA that are synthesized backward with respect to the direction of movement of the replication fork (Figure 7.3). Summary: The leading strand is synthesized continuously in the direction of replication fork movement. The lagging strand is synthesized in small pieces (Okazaki fragments) backward from the overall direction of replication. The Okazaki fragments are then joined by the action of DNA ligase