Biochem Lecture 31- DNA Replication

What is the sequence of bases in DNA encodes genetic information?

Replication → Transcription → Translation

DNA → RNA → Protein

Replication

Process of duplication of DNA (requires RNA)

Transcription

Process of formation of RNA on a DNA template (base sequence of DNA is reflected in the base sequence of RNA)

Translation

Process of protein synthesis (amino acid sequence of the protein reflects the sequence of bases in the gene that codes for that protein)

Retroviruses

Viruses in which RNA is the genetic material rather than DNA (catalyzed by reverse transcriptase)

Reverse transcriptase

Enzyme that directs the synthesis of DNA on an RNA template

Semiconservative replication

DNA replication involved in the separation of the two original strands and production of two new daughter strands using the original strands as templates

What do daughter strands contain?

Contains one template strand and one newly synthesized strand

Origin of replication

Where the DNA double helix unwinds at a specific point

How are polynucleotide chains synthesized?

In either both or in one direction from the origin of replication (biodirectional in most organisms)

Replication forks

2 at each origin of replication; points at which new polynucleotide chains are formed

Challenges in DNA replication

-achievement of continuous unwinding and separation of the two DNA strands

-synthesis of the DNA template from the 5’ to 3’ end (template has one 5’→ 3’ and one 3’ → 5’)

-guarding against errors in replication by ensuring that the correct base in added to the growing polynucleotide chains

DNA ligase

How fragments are linked together

What is DNA replication carried out by?

Replisomes

Replisomes

Complex of DNA polymerase, the DNA primer, primase and helicase at the replication form

DNA gyrase

Class II topoisomerase; catalyzes reactions involving relaxed, circular DNA with a nick in one strand to the supercoiled form with the nick sealed

Is prokaryotic DNA negatively or positively supercoiled?

Negatively; opening the helix during replication introduced positive supercoils ahead of the replication fork

What is the last nucleotide added to a growing chain?

DNA polymerase (causes the elimination of pyrophosphate and the formation of a new phosphodiester bond)

Helicase

Helix-destabilizing protein, promotes unwinding by binding at the replication fork

Single-strand binding protein (SSB)

Stabilizes single-stranded regions by binding tightly to them

primase

Enzyme that makes a short section of RNA to act as a primer for DNA synthesis

Primosome

Complex at the replication fork that consists of the RNA primer, primase and helicase

DNA polymerase

The last nucleotide to be added to a growing chain, (has a 3’-hydroxyl group on the sugar) also causes the elimination of pyrophosphate and the formation of a new phosphodiester bond

Processivity

Number of nucleotides incorporated before the dissociation of enzyme from the template

DNA Pol I

Repairs and patches DNA

DNA Pol III

Polymerization of the newly formed DNA strand (acts as a dimer) COMMENCES SYNTHESIS

DNA Pol II, IV, and V

Repairs enzymes

As the replication fork moves away ____

The RNA primer is removed by DNA Pol I and then replaced by deoxynucleotides

What is DNA ligase responsible for?

The final linking of the new DNA strand

How many times does DNA replication take place in each generation in each cell?

Only once!

Mutations

Errors in replication that occur spontaneously only once in every 10^9-10^10 base pairs and can be lethal to organisms

Proofreading

Removal of incorrect nucleotides immediately after they are added to the growing DNA during replication (only once in every 10^4-1065 base pairs)

Nick translation

Cut and patch process catalyzed by Pol I takes place during replication. Removal of RNA primer or DNA mistakes by Pol I using its 5’-3’ exonuclease activity

Mutagens

Agents that bring about a mutation that includes: ultraviolet light, ionizing radiation and various chemical agents or free radicals

Mismatch repair

Enzymes recognize that two bases are incorrectly paired, they are removed, and the area is replicated again by DNA polymerases

Base-Excision Repair

Damaged base is removed by DNA glycosylase leaving an AP site, sugar and phosphates are removed from the nucleotide by an AP endonuclease. DNA Pol I fills the gap and DNA ligase seals the phosphodiester backbone

Nucleotide-Excision Repair

Common for DNA lesions caused by UV or chemical means

Double-stranded breaks (DSB)

Breakage of both strands of a DNA molecule, poses a big threat to the stability of the genome and includes nonhomologous DNA end-joining and recombination

genetic recombination

Natural process in which genetic information is rearranged to form new associations

Homologous recombination

Involves a reaction between homologous sequences

Nonhomologous recombination

Involves combination of different nucleotide sequences

DNA recombination _____

Occurs in specific zones of the chromosome, which are called HOT SPOTS

Holiday Model

describes how recombination occurs by the breakage and reunion of DNA strands so that physical exchange of DNA parts takes place

What are the steps involved in a Holliday Model?

1- alignment of two homologous DNA segments

2- strand invasion- DNAs on the two strands swap places or crossover, at the nick

3- branch migration leads to strand exchange between the two homologous DNA pieces

In what two ways has DNA been organized in a way so it can be UNTANGLED for replication and transcription?

Supercoiling and wrapping around proteins (histones) to create chromatin

What does supercoiling in Eukaryotic DNA result in?

Chromatin!

Chromatin

Complex DNA and proteins found in eukaryotic nuclei

Histones

Basic proteins found complexed to eukaryotic DNA rich in lysine and arginine

Structure of chromatin

Beads on a string where each bead is a nucleosome and the string portions are spacer regions

Nucleosome

Globular structure in which DNA is wrapped around an aggregate of histone molecules