DNA Synthesis (Replication)

Question 1

DNA Replication in Prokaryotes

•The mechanism of DNA replication has been well studied in the
•
•Serves as a model to explain the more complex DNA replication in Eukaryotic organisms.

Answer 1

•The mechanism of DNA replication has been well studied in the bacterium E. coli.
•
•Serves as a model to explain the more complex DNA replication in Eukaryotic organisms.

Question 2

DNA Replication
•Process by which DNA is ___
•Occurs in the___ during _ phase (eukaryotes)
•Complementary parental chains act as templates for the synthesis of new strands
•____ _____ (Meselson & Stahl)
•Region where replication occurs on the DNA is known as the ___ ____
•Each new DNA molecule consists of an ___ and ___
•

Answer 2

DNA Replication
•Process by which DNA is copied
•Occurs in the nucleus during S phase (eukaryotes)
•Complementary parental chains act as templates for the synthesis of new strands
•Semi-conservative (Meselson & Stahl)
•Region where replication occurs on the DNA is known as the replication fork.
•Each new DNA molecule consists of an old and new strand.
•

Question 3

DNA Replication Requires:

• ___ ____
  
  • __ ___ and ____
  • ____involved in unwinding
• _____ ____ and ___ ___
• ___ ____

Answer 3

• Strand separation
• Coupled unwinding & synthesis
• Proteins involved in unwinding•DNA polymerases and RNA primase•DNA ligase

Question 4

Initiation of DNA Replication in Prokaryotes

• Replication begins at the ___ of ____ ( ____ )
• Ori C: ___rich region has fewer H-bonds helping to “melt” DNA
• Requires_____ ____ ____
• With the help of ____ ____ _____ ____, the DNA unwinds.
• DNA replicates ____
• Termination of replication occurs when the___ ___ ____

Answer 4

• Replication begins at the origin of replication (Ori C)
• Ori C: A-T rich region has fewer H-bonds helping to “melt” DNA
• Requires initiator protein DnaA
• With the help of Helicase, Gyrase, SSB and DnaA the DNA unwinds.
• DNA replicates bidirectionally.
• Termination of replication occurs when the two forks meet.

Question 5

Unwinding and Stabilization of DNA Strands

•DNA Helicase (_____) ___ and ____ the parental DNA strands.
•Single Strand Binding Proteins (SSB) associates with the single strands and prevents them from ____ with one another. Also protects the single strands from___ by ____
•DNA Gyrase (____) – Relieves ____ in ___of the replication fork due to unwinding of DNA by the Helicase.
•Gyrase ____ and ___ the DNA to relieve the supercoiling.
•

Answer 5

•DNA Helicase (DnaB) unwinds and separates the parental DNA strands.
•Single Strand Binding Proteins (SSB) associates with the single strands and prevents them from re-associating with one another. Also protects the single strands from cleavage by endonucleases.
•DNA Gyrase (Topoisomerase) – Relieves supercoiling in front of the replication fork due to unwinding of DNA by the Helicase.
•Gyrase cleaves and rejoins the DNA to relieve the supercoiling.
•

Question 6

Prokaryotic DNA Polymerases

•Three DNA polymerases exist in E. coli ( ___ __ ____)
•Major enzyme involved in DNA replication is ___
•Pol I is involved in ______and ________
•Pol II is involved in _____
•Pol I, II and III have ____________, while Pol ___has _________ activity as well.
•
•

Answer 6

•Three DNA polymerases exist in E. coli (Pol I, II and III)
•Major enzyme involved in DNA replication is Pol III.
•Pol I is involved in RNA primer removal and filling up the resulting gap.
•Pol II is involved in DNA repair.
•Pol I, II and III have 3’- 5’ exonuclease activity, while Pol I has 5’- 3’ exonuclease activity as well.
•
•

Question 7

DNA Polymerases III

• Require ___ templates
• Require DNA or RNA primers with a ______
• Require_____
• Possess a 3’ to 5’ exonuclease activity (Proofreading)
• Pol III has ______ – Synthesizes DNA strand ____ _____ ______from the template. (Leading strand)
• High ____ – Has ____ ___ ____in incorporating nucleotides during DNA replication.

Answer 7

• Require DNA templates
• Require DNA or RNA primers with a free 3’-OH
• Require dNTPs
• Possess a 3’ to 5’ exonuclease activity (Proofreading)
• Pol III has processivity – Synthesizes DNA strand continuously without dissociating from the template. (Leading strand)
• High fidelity – Has low error rate in incorporating nucleotides during DNA replication.

Question 8

RNA Primers are Used to Initiate DNA Synthesis

•_____ synthesizes RNA primer
___ nucleotides long
•Removed by Pol___ exonuclease activity
•Why an RNA primer?

Answer 8

• Primase synthesizes RNA primer
• ~5 nucleotides long
• Removed by Pol I exonuclease activity
• Why an RNA primer?

Question 9

Why an RNA primer is used in DNA replication?

• An enzyme that starts chains anew cannot be ____ at ____ ___
• Any enzyme that primes the synthesis of Okazaki fragments will make a relatively ____ copy (at least 1 error in ___)
• Results in ____ ____ ____
• Evolution of RNA rather than DNA for priming gives an advantage to the cell: the ribonucleotides in the primer automatically mark these sequences as “___ ____” to be efficiently removed and replaced by the 5’ to 3’ exonuclease activity of DNA pol __

Answer 9

• An enzyme that starts chains anew cannot be efficient at self-correction.
• Any enzyme that primes the synthesis of Okazaki fragments will make a relatively inaccurate copy (at least 1 error in 105)
• Results in high mutation rate.
• Evolution of RNA rather than DNA for priming gives an advantage to the cell: the ribonucleotides in the primer automatically mark these sequences as “suspect copy” to be efficiently removed and replaced by the 5’ to 3’ exonuclease activity of DNA pol I.

Question 10

DNA is Always Synthesized 5’ to 3’

•Replication fork: Site of simultaneous____and ____
•Leading strand: ___ synthesis of new DNA strand in___ direction (Pol __)
•Lagging strand: ___synthesis also in ___direction (Pol __)
•

Answer 10

•Replication fork: Site of simultaneous unwinding and synthesis
•Leading strand: continuous synthesis of new DNA strand in 5’ à 3’ direction (Pol III)
•Lagging strand: discontinuous synthesis also in 5’ à 3’ direction (Pol III)
•

Question 11

Okazaki Fragments

•Created by discontinuous synthesis on lagging strand
•Short fragments that are then ligated
_____ nt in prokaryotes
____ nt in eukaryotes
•End result is overall 5’ à 3’ synthesis
•

Answer 11

•Created by discontinuous synthesis on lagging strand
•Short fragments that are then ligated
•1-2000 nt in prokaryotes
•~200 nt in eukaryotes
•End result is overall 5’ à 3’ synthesis
•

Question 12

Trombone Model of DNA Replication

• Proteins involved in DNA replication are arranged in a ____
• Complex must move in the ____ ____
• Lagging DNA strand will ___ ___ to ensure the complex moves in the same direction.

Answer 12

• Proteins involved in DNA replication are arranged in a complex.
• Complex must move in the same direction.
• Lagging DNA strand will loop back to ensure the complex moves in the same direction.

Question 13

DNA Ligase

• RNA primers are removed and filled by DNA polymerase _. (5’ to 3’ exonuclease activity)
• DNA polymerase I can not join the ends of adjacent Okazaki fragments.
• DNA ligase ____s these adjacent Okazaki fragments together by forming an____ bond between the flanking nucleotides.

Question 14

DNA Replication: Eukaryotes
•Basic mechanism same as prokaryotes
____ polymerases but ____ ____ to E. coli DNA polymerases
•__ different DNA polymerases exist in Eukaryotic organisms.
•(pol _ – ___ ____and ____ strand synthesis, pol __– ____ strand)
•Restricted to _ phase of cell cycle
____ chromosome creates a problem – presence of telomeres can lead to___ ____

Answer 14

• Basic mechanism same as prokaryotes
• Several polymerases but analogous functions to E. coli DNA polymerases
• 16 different DNA polymerases exist in Eukaryotic organisms.
• (pol a – primase activity and lagging strand synthesis, pol d – leading strand)
• Restricted to S phase of cell cycle
• Linear chromosome creates a problem – presence of telomeres can lead to strand shortening.

Question 15

Initiation of DNA Replication in Eukaryotes

•Due to ___ OF ____ ___ eukaryotic replication forks move ___ than prokaryotic replication forks.
_____ replication from ____ origins
____ ____ _____(____) sites of ____
___ bp consensus sequence
____ ____ ____( ) contains _ proteins recognizes the____
•This complex binds to the ARS and_____ the ____

Answer 15

•Due to complexity of chromatin structure eukaryotic replication forks move slower than prokaryotic replication forks.
•Bidirectional replication from many origins
•Autonomous replication sequence (ARS) sites of initiation.
•11 bp consensus sequence
•Origin Recognition Complex (ORC) contains 8 proteins recognizes the ARS.
•This complex binds to the ARS and unwinds the DNA.
•

Question 16

The End Replication Problem

•___end of lagging strand presents unique problem.
•No place for ___ ___ to be synthesized at end of the DNA.
•Last Okazaki unit can not be synthesized.
•Different replication mechanism – Ends of DNA have unique sequences known as ____ which interacts with an enzyme.(_______)
•Telomeres - Ends of chromosomes contain _____s of ___ ____
_____ sequence
____s specific
_ _ _ _ _ _ for humans
•

Answer 16

•3’-end of lagging strand presents unique problem.
•No place for RNA primer to be synthesized at end of the DNA.
•Last Okazaki unit can not be synthesized.
•Different replication mechanism – Ends of DNA have unique sequences known as telomeres which interacts with an enzyme.(Telomerase)
•Telomeres - Ends of chromosomes contain hundreds of tandem repeats
•Hexanucleotide sequence
•Species specific
•TTAGGG for humans
•

Question 17

Telomere Replication

• Telomerase is a ___ containing ___ ____
• RNA serves as template to add ____ ____
• Recognizes the___ ____to due presence of the telomere sequence.
• Extends ____ ____strand in the 5’ to 3’ direction using the RNA template. (Adds several hexanucleotide repeats)
• These repeats provide a template for the___ and ____to synthesize the remaining Okazaki fragment.
• The extend protruding leading strand is protected from degradation by ___ a ____by specialized ____
• Loss of Telomerase leads to ___ of ___which can cause ___ ____ defects.

Answer 17

• Telomerase is a RNA containing reverse transcriptase.
• RNA serves as template to add hexanucleotide repeats
• Recognizes the leading strand to due presence of the telomere sequence.
• Extends leading DNA strand in the 5’ to 3’ direction using the RNA template. (Adds several hexanucleotide repeats)
• These repeats provide a template for the Primase and the DNA Pol III to synthesize the remaining Okazaki fragment.
• The extend protruding leading strand is protected from degradation by forming a loop by specialized proteins.
• Loss of Telomerase leads to shortening of DNA which can cause cell proliferation defects.

Question 18

Reverse Transcriptase

•Uses a single strand of____ as a template to synthesize ____
•The synthesized DNA is known as ______ or _____ ( )
______ (RNA containing virus) uses a reverse transcriptase to copy the viral ____ ____ to ____
•This DNA will____into the host genome. (Example HIV)

Answer 18

• Uses a single strand of RNA as a template to synthesis DNA.
• The synthesized DNA is known as complementary DNA or copy DNA (cDNA).
• Retrovirus (RNA containing virus) uses a reverse transcriptase to copy the viral RNA genome to cDNA.
• This DNA will integrate into the host genome. (Example HIV)

Question 19

Summary

• DNA replication is ____ and_____
• DNA polymerases are responsible for ____, ______, _____and _____
• In order to replicate the lagging strand in eukaryotic chromosomes require the addition of ____ ____ by ____

Answer 19

•DNA replication is semiconservative and semidiscontinuous.
•DNA polymerases are responsible for replication, synthesis of RNA primers, proof reading and repair of damaged DNA.
•In order to replicate the lagging strand in eukaryotic chromosomes require the addition of telomeric sequences by telomerases.
•
•