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Biochemistry chp. 20 DNA R,R,R
Review of chapter 20 (DNA Replication, Repair, and Recombination)
23
Chemistry
Undergraduate 3
12/03/2008

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Term
Replication
Definition

-Yields two identical DNA molecules ensuring transmission of information to daughter cells

- Is Semi-conservative: meaning each DNA strand serves as the template for the synthesis of a complementary strand.

Term
Basics of DNA Replication:
Definition

- Proceeds in a 5' 3' direction with the 3' OH as the point at which DNA is elongated.

- DNA strand serving as the template is read in the 3' 5' direction.

Term
DNA Polymerases (general definition)
Definition
  • Enzymes that catalyze the synthesis of DNA through a phosphory group transfer.
  • E. coli makes at least five DNA Polymerases.

 

Term
Overall reacion for all DNA synthesis
Definition

 

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Term
DNA polymerase III
Definition
  • Principal replication enzyme in E. Coli
  • At least 10 subunits
  • Fast
  • High Processivity
  • Function: chromosome Replication.
Term
Central requirements for DNA polymerization:
Definition
  • Template
    • Structure that would allow molecules to be lined up in a specific order and joined
  • Primer:
    • Strand segment (complementary to the template) with a free 3'-hydroxyl group to which a nucleotide can be added.
Term
How are growing DNA chains extended?
Definition
  • Extended at the 3' terminus
    • The nucleophile in the group transfer reaction is the 3' -OH of the primer
    • Identity of the incoming dNTP is determined by the complementary base in the template strand.
    • Proper base-pair geometry is important to fidelity.
Term
Processivity:
Definition
  • Number of nucleotides incorporated into DNA before the DNA polymerase dissociates from the primer/template.
  • Varies between a few nucleotides per binding event up to thousands.
Term
DNA Replication Accuracy:
Definition
  • Hydrogen bonding between base pairs
    • Specifies the correct pairing between complementary bases.
  • Geometry of base pairs
    • DNA Polymerase active site can only accommodate base pairs with a specific geometry.
  • Incorrect bases can be rejected before the phosphodiester bond is formed.
Term
Nuclease activities of DNA polymerases:
Definition
  • Proofreading: 3' 5' exonuclease activity.
DNAn + H2O DNAn-1 + dNMP
  • A separate active site integral to the DNA polymerase.
  • Activated by a mismatch at the 3' OH of the primer/template. Continues to act until the 3'OH is properly base-paired with the template.
  • Functions to remove nucleotides incorporated in error.
Term
Accuracy of DNA replication:
Definition
  • Accuracy of DNA synthesis: 1 error per 105 nucleotides incorporated into DNA.
  • Accuracy of DNA polymerase action (synthesis and proofreading): 1 error per 102 nucleotides incorporated into DNA.
  • Overall accuracy replication: 1 error per 109 or 1010 nucleotides incorporated into DNA.
Term
Bacterial enzymes required for chromosome replication:
Definition
  • Helicases
  • Topoisomerases
  • DNA binding proteins:
    • dsDNA binding protein (HU)
    • ssDNA binding protein (SSB)
  • Primase (DNA-dependent RNA synthase)
  • DNA polymerases I and III
  • DNA ligase (covalent joining of DNA strands)
Term
Initiation of DNA replication in E. coli:
Definition
- Formation of the theta structure or replication bubble occurs at the E. coli. replication origin with establishment of two replication forks.
 
 
Term
Primosome:
Definition
  • Complex of DnaB*DnaG.
  • Functions to:
    • Unwind the parental duplex ahead of the DNA polymerase.
    • Synthesize RNA primers (primase enzyme)
Term
Nick translation:
Definition
- Process in which DNA polymerase binds to a gapbetween the 3' end off a nascent DNA chain and the 5' end of the next RNA primer, catalyzes hydrolytic removal of ribonucleotides using 5' 3' exonuclease activity, and replaces them with deoxyribonucleotides using 5' 3' polymerase activity.
Term

[Chromosome replication in bacteria]

Termination:

Definition
  • Meeting of two replication forks.
  • Resolution of the two linked circles.
  • Segregation of daughter chromosomes into separate cells.
Term

[Eukaryotic DNA Polymerases]

DNA polymerase δ:

Definition
  • Replicates the leading and lagging strands of the fork.
  • Contains both polymerase and 3' 5' exonuclease activities.
  • Employs a "sliding clamp" protein called PCNA, which ensures high processivity. PCNA is functionally homologous to the β subunit of DNA polymerase III
Term

[Eukaryotic DNA Polymerases]

DNA polymerase α:
Definition
  • Synthesizes RNA primers for the lagging strands of the fork.
  • Contains DNA and RNA polymerase activities on separate subunits.
  • Has no 3'-5' or 5'-3' nuclease activities.
Term

[Eukaryotic DNA Polymerases]

DNA polymerase ε:
Definition
  • May remove primers by nick translation.
  • Contains DNA polymerase activity
  • Has both 3'-5' and 5'-3' nuclease activities.
Term
Mutations:
Definition

- Any permanent heritable change in the sequence of genomic DNA.

 

  • Substitution mutation: replacement of one base pair with another.
  • Insertion or deletion mutations: addition or deletion of one or more base pairs.
Term
Mutagens:
Definition
  • Deaminating agents
  • Base analogs
  • UV radiation
  • Alkylating agents
  • Hydroxylating agents
  • Intercalating agents
  • Ionizing radiation (x-rays, gamma-rays)
  • Transposable elements including retroviruses
  • DNA replication / cell division
Term
Base Excision Repair:
Definition
  • Lesions (unrepaired DNA) that are repaired by this pathway include:
    • Base deamination( e.g. cytosine deamination to yield uracil)
    • Oxidized and alkylated bases
    • Spontaneous loss of a base.
  • DNA glycosylases specific for a particular lesion hydrolyze the N-glyosidic bond. (e.g. Uracil DNA glycosylase)
  • An apurinic or apyrimidinic (AP) site is created as the first step in repair.
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