DNA and the Cell Cycle › DNA Replication

DNA Replication Part I

Notes

DNA Replication Part I

Sections

DNA Replication

  • Complementary
  • Semiconservative
  • Bidirectional
  • Occurs in the S phase
  • Synthesis in the 5' to 3' direction – antiparallel

KEY ENZYMES

  • Helicase separates DNA strands at replication fork
  • Topoisomerase Relieves supercoiling downstream of replication fork
  • Primase synthesizes RNA primer
  • DNA polymerase synthesizes DNA
  • Ligase joins DNA fragments together

Replisome complex

  • Protein complex assembled at each replication fork
  • Comprises: helicase, primase, DNA polymerases, single-strand binding proteins

PROKARYOTIC REPLICATION (E. COLI)

  • DNA is melted at one origin of replication (region rich in adenine and thymine)
  • 2 replication forks with replisomes on each side of origin are created (bidirectional replication)
  • Helicase separates parent DNA strand (breaks H-bonds)
  • Single-stranded DNA binding proteins stabilize strands (prevent degradation)
  • Primase synthesizes RNA primers (~ 10 nucleotides long)
  • DNA polymerase elongates strand (adds to 3' end only)
  • Leading strand: oriented 3' to 5' towards fork
  • Lagging strand: oriented 5' to 3' towards fork --> Okazaki fragments (discontinuous replication)
  • Different DNA polymerase removes primer & fills gap with DNA
  • DNA ligase joins Okazaki fragments
  • Topoisomerase relieves supercoiled DNA downstream of replication fork

DNA Directionality

  • Synthesized 5' to 3' direction but read in the 3' to 5' direction
  • Produces antiparallel but complementary DNA strands
  • Leading strand: DNA synthesis continuous and towards replication fork
  • Lagging strand: DNA synthesis discontinuous and away from replication fork (Okazaki fragments)

CLINICAL CORRELATION

AZT

  • Anti-HIV drug that blocks viral DNA replication
  • Adds to daughter strand during viral replication and prevents elongation

Full-Length Text

  • Here we will learn the process of DNA replication.
    • This is part one of a two part tutorial.
    • In part one we use E. Coli as a model for prokaryotic DNA replication.
    • In part two, we learn the similar, but slightly different, process of eukaryotic DNA replication.
  • First, start a table to summarize some key points about DNA replication (in both prokaryotes and eukaryotes).
  • Denote the following:
    • DNA replication occurs in the S phase of the cell cycle. (S for DNA Synthesis)
    • It is semiconservative, each DNA strand is a template for its own complementary strand.
    • It is bidirectional, which we will explain shortly.

Now, let's illustrate how DNA replication is initiated in prokaryotes, specifically E. coli.

  • Draw a circular, double stranded DNA molecule with a large bubble in the middle.
  • Label the bubble the "origin of replication." E. coli has a single origin of replication.
  • Label each side of the bubble "replication fork."
  • Use arrows to indicate that replication begins at each of the two replication forks and continues in opposite directions.
    • This is what "bidirectional replication" means; replication proceeds in two different directions, simultaneously.

Now, before we move on, let's first identify the key molecules involved in DNA replication.

  • Denote that both prokaryotic and eukaryotic DNA replication involve the following:
    • Helicase, an enzyme that catalyzes DNA strand separation.
    • Topoisomerase, which alleviates the super-coiling of DNA downstream of the origin.
    • Primase, which synthesizes RNA.
    • DNA polymerase, which synthesizes DNA. There are multiple DNA polymerases, each with a specialized function.
    • Ligase, which ligates fragments of DNA together during replication.
  • Imagine that all of these enzymes (except for topoisomerase and ligase) are clamped together in one large machine that slides along both parent strands.
  • Write that this machine is called a replisome, and that it assembles at each replication fork.
    • We will leave the clamp and the clamp-loading proteins out of our diagram for clarity. (Again, topoisomerase and ligase are not part of the replisome. This will become clear later.)

Now, let's return to our origin of replication.

  • Write that it is rich in adenine–thymine base pairs and that specialized proteins recognize this region and "melt" the parental strands, which initiates replication.

Now, let's zoom in on one of the replication forks.

  • Draw a replication fork and label the 5-prime and 3-prime ends of each parent strand.
  • Show that helicase separates the parent DNA strands by breaking hydrogen bonds, which creates an origin of replication.
  • Illustrate that single-stranded DNA binding proteins stabilize the strands and protect them from degradation.
  • Now, show that topoisomerase relieves supercoiled DNA downstream of the replication fork.
    • Thus, it is not part of the replisome, which is localized at the replication fork.

To visualize this draw a loosely coiled jump-rope.

  • Show that if you pick up the handles and pull them apart, it supercoils the rest of the jump-rope.
  • Show that topoisomerases alleviate this tension by breaking and rejoining DNA strands. We describe their mechanism in detail elsewhere.

Now, let's discuss the synthesis of DNA.

  • First, write that DNA is synthesized in a 5-prime to 3-prime direction, antiparallel to the parent strand.
    • DNA polymerase can only add nucleotides to free 3-prime hydroxyl groups; thus, it can only replicate DNA in the 5-prime to 3-prime direction.
  • Next, label the parent strand that is oriented as 3-prime to 5-prime (towards the fork) as the "leading strand."
  • Label the remaining parent strand (which is oriented 5-prime to 3-prime) as the "lagging strand."
  • Now, illustrate leading strand replication by drawing a complementary newly synthesized strand.
  • Label the 5-prime and 3-prime ends of this daughter strand.
  • At the 5-prime end of the daughter strand, draw a short RNA primer.
  • Indicate that primase synthesizes this short, complementary RNA primer (about 10 nucleotides long), which facilitates the addition of nucleotides by DNA polymerase.
  • Show that DNA polymerase copies the parent strand by catalyzing the addition of nucleotides to the 3-prime end of the daughter strand.

Now, let's draw the primer on the lagging strand.

  • Draw a primer complementary to the 3-prime end of the lagging strand.
    • The 3-prime end of the lagging strand provides the template for the 5-prime end of the daughter strand.
  • Show that DNA polymerase synthesizes a new, complementary daughter strand as it moves away from the replication fork.
  • As helicase continues to unzip the parent strands downstream, new primers must be added to replicate newly exposed DNA.
    • To suggest this, draw another primer on the lagging strand.
  • Draw another fragment of complementary, newly synthesized DNA.
  • Label these fragments Okazaki fragments.
  • Write that replication of the lagging strand is discontinuous.
    • DNA polymerase copies the parent strand until it runs into the RNA primer of a previously synthesized Okazaki fragment.
  • Indicate that a different DNA polymerase removes the primer and fills the gap with DNA (instead of the original RNA).
  • Show that DNA ligase joins the Okazaki fragments together.
    • Thus, the lagging strand "lags" behind the leading strand because it requires these extra steps.
  • Note that the molecules involved in these extra steps are not part of the replisome machine because they act upstream of the replication fork.
  • As a clinical correlation, denote that AZT is an anti-HIV drug that blocks viral DNA replication.
    • During viral DNA replication, AZT adds to the daughter strand and prevents it from elongating further.

In part II, we will address DNA replication in eukaryotes, highlighting key differences between eukaryotic and prokaryotic process. We will also discuss DNA polymerase function in more detail.

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