DNA Replication and Repair Flashcards

DNA Replication and Repair

Problems During DNA Replication

Every cell must address these challenges during DNA replication:

• Strand Polarity: DNA strands are antiparallel, with one strand running 5' to 3' and the other 3' to 5'.
• Unzipping: The double helix must be unwound to allow access for DNA polymerase.
• Processivity: Maintaining contact between DNA polymerase and the DNA strand for long stretches of synthesis.
• Untangling: Unwinding DNA can lead to tangling and supercoiling.
• Copy Number Control: Ensuring only one copy of DNA is made per cell division cycle.
• Accuracy: Minimizing mistakes during the copying of millions/billions of nucleotides, as errors can lead to mutations.

Coordinating DNA Synthesis

• Both DNA strands are copied simultaneously. However, due to the antiparallel nature, different mechanisms are required.
• Leading Strand: Synthesized continuously in the 5' to 3' direction.
• Lagging Strand: Synthesized discontinuously in short fragments (Okazaki fragments) because it appears to be replicating in the "wrong" direction (3' to 5').
• The same DNA polymerase is used for both strands, but it can only synthesize in the 5' to 3' direction.
• Unzipping, processivity, and untangling occur concurrently with DNA synthesis.

Evolutionary Conservation

• The mechanisms and machinery for DNA replication are highly conserved between bacteria and eukaryotes.
• Much of our understanding of DNA replication mechanisms comes from studying bacteria.

Eukaryotic vs. Prokaryotic Replication

Eukaryotes have unique challenges:

• Linear Chromosomes:

  • The five prime end of a linear DNA strand cannot be completely duplicated by DNA polymerase, leading to potential shortening of the chromosome with each division.
  • Linear chromosomes require specialized structures at the ends (telomeres) to distinguish them from broken DNA molecules.

• Telomeres: Prevent chromosome shrinkage during replication and ensure that the cell recognizes the chromosome ends as intact.

• Histones:

  • Eukaryotic DNA is packaged with histones, which must be duplicated along with the DNA.
  • Histone modifications (methylation, acetylation, etc.) play a role in gene regulation and must also be duplicated during replication.

Accuracy of Replication

• DNA replication occurs with astonishing accuracy, about one mistake per 10^{10} nucleotides.
• Additional mechanisms exist to repair DNA damage from environmental factors, separate from the error correction during replication.

Semi-Conservative Replication

• Each new DNA molecule consists of one parental strand and one newly synthesized strand. This is called the semi-conservative mechanism.
• If a mutation occurs during replication, it will be copied in subsequent generations.

DNA Polymerase Mechanism

• DNA polymerase synthesizes exclusively in the 5' to 3' direction, adding new nucleotides to the existing 3' end.
• Deoxynucleotide triphosphates provide the energy for polymerization through the hydrolysis of two phosphates.
• DNA polymerase cannot initiate a new strand; it requires an existing chain (primer).
• An RNA primase synthesizes a short RNA primer to provide the free 3' hydroxyl group needed for DNA polymerase to begin synthesis.
• DNA replication occurs in the five prime to three prime direction; the new nucleotide, starting as a nucleotide triphosphate, is added to the three prime end of our growing strand of DNA.
• DNA polymerase cannot initiate polymerization and can only add a nucleotide to an existing strand.
• RNA primase synthesizes an RNA primer, which remains attached to the DNA strand and provides the three prime hydroxyl end for adding the first DNA nucleotide.

DNA Polymerase Structure and Function

• DNA polymerase has a "hand" structure with fingers, a palm, and a thumb.
• The DNA template sits in the palm, and the thumb holds it in place.
• DNA polymerase adds an incorrect nucleotide approximately once every 10^5 nucleotides.

Addressing Replication Problems

The DNA polymerase complex addresses the strand polarity, unzipping, and processivity problems simultaneously:

• Strand Polarity: Two DNA polymerase molecules are used (one for each strand). The lagging strand is synthesized in Okazaki fragments.
• Unzipping: DNA helicase unwinds the double helix.
• Processivity: A sliding clamp keeps the DNA polymerase in contact with the DNA.

Leading vs. Lagging Strand

• The two DNA polymerase molecules are physically attached.
• The leading strand is synthesized continuously in the 5' to 3' direction.
• The lagging strand requires a loop to allow the DNA polymerase to synthesize in the 5' to 3' direction overall but appears to be traveling backwards on the DNA strand.
• This loop results in discontinuous synthesis of Okazaki fragments.
• Each Okazaki fragment requires a new RNA primer.

Okazaki Fragments Processing

• Primers are removed.
• DNA repair polymerase fills in the gaps.
• DNA ligase seals the fragments.

Unzipping the DNA

• DNA helicase unwinds the DNA by hydrolyzing ATP, causing the complex to rotate and pry apart the DNA strands.
• Single-stranded DNA-binding proteins (SSBPs) prevent the DNA from re-annealing behind the helicase.

Processivity

• A sliding clamp maintains contact between the DNA polymerase and the DNA.
• A clamp loader uses ATP to load the clamp onto the DNA.

Replication Complex

• Includes two DNA polymerase molecules, DNA helicase, sliding clamps, and a clamp loader.
• In eukaryotes, the helicase is on the template for the leading strand. (Note: In prokaryotes, the helicase is on the template for the lagging strand.)
• The leading strand follows the helicase, while the lagging strand forms a loop to allow synthesis in the correct direction.

Untangling DNA

• Unwinding DNA introduces torsional stress, leading to supercoiling and tangling.
• Topoisomerases relieve this stress by cutting and rejoining DNA strands.

Topoisomerases

• Topoisomerase I:
  • Uses a "nick and swivel" mechanism.
  • Tyrosine residue in the enzyme breaks a phosphodiester bond in one DNA strand.
  • Allows strands to rotate and relieve stress.
  • Rejoins the strand without ATP hydrolysis (energetically neutral).
• Topoisomerase II:
  • Forms a homodimer that makes a double-stranded cut in the DNA.
  • Passes another DNA double helix through the break.
  • Seals the break, requiring ATP hydrolysis.

Differences Between Topoisomerases

• Topoisomerase II is faster at removing supercoils and can unlink tangled DNA strands.
• Topoisomerase I is more energy-efficient when continuously unwinding ahead of the helicase because it does not require ATP.
• Topoisomerase II is needed to unlink the two rings of DNA that arise from duplication of a circular chromosome.

Origin of Replication

• DNA replication initiates at specific sites called origins of replication.
• Bacterial chromosomes have a single origin, whereas eukaryotic linear chromosomes have multiple origins.
• Origins of replication are AT-rich regions, which are easier to separate because A-T base pairs have only two hydrogen bonds vs three for G-C base pairs.

Replication Forks and Bubbles

• Helicases open the DNA at the origin, forming a replication bubble.

• Each bubble has two replication forks.

• RNA primers are synthesized on both strands, initiating leading and lagging strand synthesis.

• Four DNA polymerase molecules are present in each replication bubble: two moving left, two moving right.

  Bacterial Replication Fork

• Initiator proteins bind to the origin of replication.

• Helicase binds to unwind the DNA.

• DNA primase synthesizes the RNA primer.

• The leading strand can now be synthesized.

• Two replication forks move in opposite directions.

• Topoisomerase II separates the two circular DNA molecules upon completion of replication.

Preventing Re-replication in Prokaryotes

• After replication starts, there are two origins of replication.
• The cell must prevent re-replication.
• Within the origin of replication, there is a repeated four-nucleotide motif, GATC, which is a palindrome.
• DNA adenine methylase (Dam methylase) methylates the As in the origin of replication.
• DNA can be initiated only if it is methylated on both strands.
• After replication, the newly synthesized strand is not methylated, forming hemimethylated DNA.
• Dam methylase is not expressed until replication is complete.
• This creates a refractory period, preventing re-replication; mutation on the damn methylase will enable replication to occur before DNA is fully duplicated.

Preventing Re-replication in Eukaryotes

• Eukaryotic DNA replication is synchronized with the cell cycle, and regulation is more complex.
• They have many origins of replication along the DNA.

Single Replication control

• The origin recognition complex (ORC) binds to the origin of replication.
• Accessory proteins (Cdc6 and Cdt1) bind to the ORC and recruit helicase.
• This occurs during the G1 phase, forming the pre-replicative complex.
• The helicase is inactive at this stage.
• Progression to the S phase is controlled by cyclin-dependent kinases (cdks).
• Cdk phosphorylates Cdc6, causing it to degrade and dissociate the helicases to start synthesis.
• In the absence of Cdc6, a new replicative complex cannot be formed.
• CdK also phosphorylates the ORC, preventing further recruitment of Cdt1 and Cdc6.
• At the end of mitosis, the kinase is removed, and a phosphatase re-establishes the pre-replicative complex in G1.

Histone Duplication

• Chromatin structure (histones) must be duplicated during DNA replication.
• Histones two a and two b are removed from the DNA during replication, but histones three and four remain attached.
• Histones three and four are randomly distributed between the two daughter strands.

Histone Replication challenges

• Histones 2A and 2B are removed from the DNA during replication.
• Histones 3 and 4 remain attached to the DNA but are randomly distributed between the two daughter strands, histone chaperones loaded as well.

Duplicating the Histone Code

• The cell assumes that the same code must be used in any location where the histone code exists.
• A reader-writer complex reads the histone code on the existing histones three, four complexes, and copies them onto the newly added histones.

The End Replication Problem

• In linear chromosomes, the five prime end cannot be completely replicated because there is no template to extend an RNA primer to fill the gap left after primer removal.

Telomeres

• Counteract this problem in embryonic cells, stem cells, and certain immune cells.
• Telomerase, a reverse transcriptase (RNA-dependent DNA polymerase), synthesizes telomeres, repetitive sequences at the ends of chromosomes.
• Telomerase extends the three prime end of the template strand, providing a template for filling in the 5 prime end of the daughter strand.
• Cells lacking telomerase have a limited number of replications before the telomeres shorten to a critical length; This is called the Hayflick limit.
• Cancer cells often express telomerase, allowing them to divide indefinitely.

Error Correction Mechanisms

• DNA polymerase has a proofreading mechanism that corrects errors during replication, which involves checking the most recently added nucleotide.
  DNA polymerase accuracy is about 1 in 10^5 , so the fidelity of replication must be increased.

Error Correction Challenges

• RNA molecules are not kept in the cell, making mistakes in RNA not devastating as mistakes in the DNA.
• Basic polymerase has the mutation rate of one in 10^5.

Proofreading Mechanism of DNA Polymerase

• DNA polymerase has is a type of secondary mechanism where it checks if the three prime most nucleotide is correctly paired
• the pair bond must be correct and if it isnt the it will remove the three prime most nucleotide and start all over
• 99 percent of erorrs are caught by this proofreading mechanism
• That proofreading mechanism means that the DNA Polymerase will not contine until there base pair is correct
• if you dont hace a primmer there is no three prime nucleotide to check for proof reading
• In order to have a primer, it costs 20 nucleotides worth of ATP to ensure the DNA strand is increasted in accuracy by 100 fold.
• the end replication problem that you cant synthesize in the three prime to five prime direction, and you need a primer in order to synthesize, those stem from the proofreading mechanism that evolved in bacteria with circular chromosomes. If you have a circular chromosome, neither of those is an issue, and so evolving this mechanism would be easy. In eukaryotes, that mechanism now adds the end replication problem.

Problems with Erros, Error corection on the three prime nucleotide not checking

• there isnt any proof reading
• DNA polymerase will miss the eror and move on
• the error repair mechanism ican not determine which strand is correct and which is incorrect
• there are errors due to DNA damage as well

DNA directed Mismatch repairs

• Strand must be parent strand vs the non parent strand to be able to correct errors
  Strand directed mismatch repair detects mutation errors from the DNA with the strand is seperated in that area. you get a distortion
  Replicative damage from dna synthesis you can remove incorrect nucleotides
  you can not determine which of the mutant stand and the correct strand after. you must use a parental and non parental

Mismatch correction

• After the error is fixed, the cell moves on.
  This mechancalism works by having mutS and MutL for example bind
  this bind and scans until it finds a unsealed nick, there is an un sealed nick due to the synthesis of dNA having RNA Primer still. then there will be another nick the the original erron dna that will be removed used using dna repair polymerase ligase will come and add a neck, so basically parental non parnetal vs methylation
• if there are a methylated strand you will be able yo detect the strand to figure out the right dna that the cell will use.
• If Damage acctual occurs there not mechanism to know which to fix