DNA Replication
DNA Replication DNA Structure • Double Helix • Anti-parallel • 5’ à 3’ • Complimentary base pairing = ______ & _________ • Purines pair with Pyrimidines
How to remember • CUT the Py à Cytosine, Uracil, Thymine are Pyrimidines • Pure As Gold à Purines are Adenine and Guanine Making the DNA Backbone • Sugar-phosphate backbone is made of _______________ bonds • Are made through ____________ • DNA polymerases, Ligase, Topoisomerase, Telomerase can make these bonds
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DNA Replication – The Replisome • Replisome = all the DNA, RNA and proteins involved at a replication fork/bubble • Helicase – Opens the double stranded DNA and unwinds it in both directions • Single-stranded binding protein – bind single-stranded DNA to make sure they do not re-anneal • Topoisomerase – works ahead of the replication fork to decrease supercoiling of the DNA caused by helicase unwinding o Makes a single stranded cut in the backbone o Makes a temporary phosphodiester bond between itself and the cut strand o Feeds the uncut strand through the cut o Reforms the phosphodiester bond on the cut strand • DNA Primase – Uses complimentary base pairing to make RNA primers • DNA polymerase alpha – begins eukaryotic DNA replication by extended the 3’ end of the RNA primer for about 40 base pairs until polymerase switching occurs • DNA polymerase delta/epsilon – extends the 3’ end of the nucleotide sequence made by DNA polymerase alpha, delta on the leading strand, epsilon on the lagging strand to make Okazaki fragments • RNase H/Flap endonuclease – remove RNA primers • DNA Ligase – makes phosphodiester bonds between the nicks in the DNA backbone Replication Bubbles • Eukaryotes have up to 100,000 origins of replication o Replication bubbles extend in both directions until they meet the next replication bubble • Bacteria (they have circular chromosomes) have 1 origin of replication Draw a Replication Bubble
DNA Polymerase Proofreading • DNA polymerase is an exonuclease • Exonuclease = breaks phosphodiester bonds from the end of a DNA strand
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Endonuclease = breaks phosphodiester bonds anywhere along the DNA backbone
Telomerase
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Expression of Telomerase • Telomerase is only expressed in highly proliferative cells • Embryonic stem cells • Sperm cells • Some adult stem cells • Cancer cells • In adult somatic cells telomere length shortens with each cell division • When telomeres reach a critical length à the cell __________
DNA Replication: Summary • • • • • •
DNA replication occurs in ___ phase of the cell cycle Replication is ______-conservative = Leading strand is replicated _______________, lagging strand is replicated __________________ DNA polymerase needs an _______ primer to start replication DNA polymerase can ONLY extend from __________ The _____ ends of the chromosomes are extended by _____________ • There extended ends are called _____________ Telomeres get shorter with each replication in somatic cells
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Practice Problems You provide a eukaryotic cell with radioactively labeled cytosine during S phase of the cell cycle. If you were to visualize this cells chromosomes in a metaphase spread, where would the radioactive cytosine be? a) There would be no incorporation of radioactive cytosine as it would be removed through DNA polymerases proof-reading mechanism b) It would be in all sister chromatids c) It would be in only 1 of 2 sister chromatids d) It would only be on one arm of the chromosome DNA replication is referred to as semi-conservative or semi-continuous, what does that mean? a) That the ends of chromosomes shorten with each cell division b) DNA polymerase sometimes to has backup to replace a miss-paired base c) DNA is replicated as one long strand on the leading strand and as Okazaki fragments on the lagging strand d) The replication bubble continues in both directions Which of the following about DNA replication is true? 1. The same template DNA is responsible for replicating the entire leading strand chromosome 2. Replication bubbles in bacteria and eukaryotes are very different 3. The same DNA polymerase replicates both the leading and lagging strands 4. Telomerase needs an RNA primer made by primase to extend telomeres a) 1, 2 and 3 only b) 1 and 3 only c) 4 only d) All are correct e) None are correct
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How to remember • CUT the Py à Cytosine, Uracil, Thymine are Pyrimidines • Pure As Gold à Purines are Adenine and Guanine Making the DNA Backbone • Sugar-phosphate backbone is made of _______________ bonds • Are made through ____________ • DNA polymerases, Ligase, Topoisomerase, Telomerase can make these bonds
© Wizedemy Inc.
DNA Replication – The Replisome • Replisome = all the DNA, RNA and proteins involved at a replication fork/bubble • Helicase – Opens the double stranded DNA and unwinds it in both directions • Single-stranded binding protein – bind single-stranded DNA to make sure they do not re-anneal • Topoisomerase – works ahead of the replication fork to decrease supercoiling of the DNA caused by helicase unwinding o Makes a single stranded cut in the backbone o Makes a temporary phosphodiester bond between itself and the cut strand o Feeds the uncut strand through the cut o Reforms the phosphodiester bond on the cut strand • DNA Primase – Uses complimentary base pairing to make RNA primers • DNA polymerase alpha – begins eukaryotic DNA replication by extended the 3’ end of the RNA primer for about 40 base pairs until polymerase switching occurs • DNA polymerase delta/epsilon – extends the 3’ end of the nucleotide sequence made by DNA polymerase alpha, delta on the leading strand, epsilon on the lagging strand to make Okazaki fragments • RNase H/Flap endonuclease – remove RNA primers • DNA Ligase – makes phosphodiester bonds between the nicks in the DNA backbone Replication Bubbles • Eukaryotes have up to 100,000 origins of replication o Replication bubbles extend in both directions until they meet the next replication bubble • Bacteria (they have circular chromosomes) have 1 origin of replication Draw a Replication Bubble
DNA Polymerase Proofreading • DNA polymerase is an exonuclease • Exonuclease = breaks phosphodiester bonds from the end of a DNA strand
© Wizedemy Inc.
•
Endonuclease = breaks phosphodiester bonds anywhere along the DNA backbone
Telomerase
1
2
3
4
5
6
7
8
9
© Wizedemy Inc.
Expression of Telomerase • Telomerase is only expressed in highly proliferative cells • Embryonic stem cells • Sperm cells • Some adult stem cells • Cancer cells • In adult somatic cells telomere length shortens with each cell division • When telomeres reach a critical length à the cell __________
DNA Replication: Summary • • • • • •
DNA replication occurs in ___ phase of the cell cycle Replication is ______-conservative = Leading strand is replicated _______________, lagging strand is replicated __________________ DNA polymerase needs an _______ primer to start replication DNA polymerase can ONLY extend from __________ The _____ ends of the chromosomes are extended by _____________ • There extended ends are called _____________ Telomeres get shorter with each replication in somatic cells
© Wizedemy Inc.
Practice Problems You provide a eukaryotic cell with radioactively labeled cytosine during S phase of the cell cycle. If you were to visualize this cells chromosomes in a metaphase spread, where would the radioactive cytosine be? a) There would be no incorporation of radioactive cytosine as it would be removed through DNA polymerases proof-reading mechanism b) It would be in all sister chromatids c) It would be in only 1 of 2 sister chromatids d) It would only be on one arm of the chromosome DNA replication is referred to as semi-conservative or semi-continuous, what does that mean? a) That the ends of chromosomes shorten with each cell division b) DNA polymerase sometimes to has backup to replace a miss-paired base c) DNA is replicated as one long strand on the leading strand and as Okazaki fragments on the lagging strand d) The replication bubble continues in both directions Which of the following about DNA replication is true? 1. The same template DNA is responsible for replicating the entire leading strand chromosome 2. Replication bubbles in bacteria and eukaryotes are very different 3. The same DNA polymerase replicates both the leading and lagging strands 4. Telomerase needs an RNA primer made by primase to extend telomeres a) 1, 2 and 3 only b) 1 and 3 only c) 4 only d) All are correct e) None are correct
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