D1.1 DNA replication

What are the main enzymes involved in DNA replication and what is its function?

1) DNA helicase

• unwinds double helix structure

• break hydrogen bonds holding double strands together

2) DNA polymerase

• reads old strands as a template to synthesize new strand

• adds free nucleotides to the new strand using complementary base pairing

what are some properties of DNA replication

• produces identical DNA copies (with identical base sequence)

• semi-conservative (each DNA copy contains one old strand, one new strand)

• relies on complementary base pairing

  • nucleotide selectivity of DNA polymerase

  • high degree of accuracy to maintain in genetic integrity

  • allows for genetic continuity

what happens in polymerase chain reaction (PCR)?

• amplifies small amounts of DNA

  • relies on DNA replication

• each cycle doubles the amount of DNA (usually run for 30 cycles)

• performed in the lab under specific conditions

what are the stages in polymerase chain reaction

• 90^oc to separate double stranded DNA and cooled to 55^oc

• primers (a short DNA sequence that initiates replication) are binded to DNA strands

• 75^oc for Taq polymerase to synthesize new strand starting from the primers

what is gel electrophoresis?

a process to separate DNA fragments based on size

how does gel electrophoresis work?

• DNA is negatively charged so it moves from negative to positive electrode

• shorter fragments move faster and larger, longer fragments moves slower

How is DNA profiling used to identify an individual?

• DNA profiling is used in criminal investigations and paternity testings

• individuals have unique genetic markers that contain varying numbers of STR (short tandem repeats)

• polymerase chain reaction to amplify initial DNA sample collected

• enzymes used to cut DNA at certain DNA markers to create fragments

• gel electrophoresis to separate fragments and create DNA profile

In what direction does DNA polymerase work in?

DNA polymerase adds new nucleotides by attaching 5’ of a DNA nucleotide to the 3’ end of a strand

what are the enzymes involved in DNA replication and what are their functions.

• DNA helicase

  • breaks hydrogen bonds between strands, creating replication fork

• DNA primase

  • adds a primer (short RNA sequence) to each strand

    • indicates a starting point for DNA polymerase

• DNA polymerase III

  • adds 5’ end of new nucleotides to the 3’ end of the new strand

why are primers needed for DNA replication?

DNA polymerase cannot initiate replication and can only add nucleotides to an existing strand

describe how DNA is replication on the leading strand and the lagging strand.

In the leading strand, DNA polymerase III moves towards replication fork continuously.

In the lagging strand, DNA polymerase III moves away from replication fork in fragments, and it is discontinuous.

what are the short sequences of DNA on the lagging strand called?

okazaki fragments

what is the function of DNA polymerase I?

removes primers and replaces with DNA nucleotides

what is the function of DNA ligase?

joins Okazaki fragments together with phosphodiester bonds

describe in detail the replication process on the lagging strand

• DNA polymerase moves from 5’ to 3’

• It moves away from the replication fork

• DNA polymerase III has to return back to rhe replication fork every time

• therefore, short fragments called okazaki fragments (each with a primer) is added on the lagging strand

• DNA polymerase I replaces primers with DNA nucleotides

• DNA ligase joins the fragments together

what enzyme is in charge of DNA proofreading and why is it important?

• DNA polymerase III

• it is important because…

  • approximate error rate of 1 mismatched base every 10¹⁰ nucleotides

  • DNA polymerase III will “proofread” and then “excise” a mismatched base from the 3’ terminal of strand and replace with correct nucleotide