Genetics - Lecture 10: DNA, RNA, Replication, and Transcription

Semiconservative Replication

• All DNA replication is this type

• Replication in which the two nucleotide strands of DNA separate, and each serves as a template for the synthesis of a new strand.

  • one round of replication would produce two hybrid molecules, each consisting of half original DNA and half new DNA

• There are several different ways semiconservative replication can take place, depending on the template DNA—that is, whether it is linear or circular.

• Called this because original nucleotide strands remain conserved, the original DNA molecule is half (semi-) conserved during replication.

Replicon

Unit of replication consisting of DNA from the origin of replication to the point at which replication on either side of the origin ends

Origin of replication

Site where DNA replication is initiated

Theta Replication

• Replication of circular DNA (E. coli, bacteria, etc.) that is initiated by the unwinding of the two nucleotide strands, producing a replication bubble

• Unwinding continues at one or both ends of the bubble

• DNA replication on both template strands is simultaneous with unwinding until the two replication forks meet

Bidirectional replication

If two replication forks proceed outward in both directions - bidirectional replication - unwinding and replicating until they meet.

Unidirectional Direction.

Occurs with a single replication that proceeds around the entire circle

Requirements of replication (linear eukaryotic replication)

1. A template consisting of single-stranded DNA

2. Raw materials (substrates) to be assembled into a new nucleotide strand.

• The raw materials from which new DNA molecules are synthesized are deoxyribonucleoside triphosphates (dNTPs), each consisting of a deoxyribose sugar and a base (a nucleoside) attached to three phosphate groups.

3. Enzymes (e.g. polymerase) and other proteins that “read” the template and assemble the substrates into a DNA molecule.

Direction of Replication

• synthesizesd 5’ to 3’ by DNA polymerase

DNA synthesis requires…

Enzymes and proteins

DNA Polymerase

Enzymes that synthesizes DNA.

• Only adds nucleotides to the 3′ end of the growing strand (not the 5′ end)

• New DNA strands always elongate in the same 5′ to 3′ direction

DNA Synthesis

opposite directions on two DNA template strands.

• As DNA unwinds during replication, one template is exposed in the 5′ → 3′ direction, the other in the 3′ → 5′ direction.

Leading Strand

DNA strand that is replicated continuously

Continuous Replication

Replication of the leading strand of DNA in the same direction as unwinding, allowing new nucleotides to be added continuously to the 3’ end of the new strand as the template is exposed

Lagging Strand

DNA strand that is replicated discontinuously

Discontinuous Strand

- Replication of the lagging strand of DNA in the direction opposite of unwinding, which means that DNA must be synthesized in short stretches (Okazaki fragments)

Okazaki Fragments

Short length of newly synthesized DNA produced by discontinuous replication on the lagging strand; these fragments are eventually joined together

4 stages of Bacterial DNA replication

1. Initiation

2. Unwinding

3. Elongation

4. Termination

Initiation

• begins at a single origin of replication = oriC

• in bacteria there is only one origin of replication

• uses initiator proteins

Initiator proteins

Protein that binds to an origin of replication (OriC) and causes a short section of DNA to unwind, allowing helicase and other single-strand-binding proteins to attach to the polynucleotide strand

• DnaA in E. Coli

Unwinding

Uses:

1. DNA Helicase

2. Single-strand-binding (SSB) Proteins

3. DNA Gyrase

DNA Helicase

Enzyme that unwinds dsDNA by breaking hydrogen bonds that exist between the bases of the two nucleotide strands of a DNA molecule

Single-strand-binding (SSB) Proteins

Protein that attaches to the exposed ssDNA during replication and prevents formation of secondary structures that would interfere with replication

DNA Gyrase

Topoisomerase enzyme in E. coli that relieves the torsional strain that builds up ahead of the replication fork

Elongation

single-stranded DNA used as template for synthesis of DNA

• uses:

  • Primers

  • Primase

  • DNA Polymerase III

  • DNA Polymerase I

  • DNA Ligase

Primase

Enzyme that synthesizes a short stretch of RNA on a DNA template; in replication it provides a 3’-OH group for the attachment of a DNA nucleotide

Primers

Short stretch of RNA on a DNA template; provides a 3’-OH group for the attachment of a DNA nucleotide at the initiation of replication.

• 10 -12 nucleotides long

DNA Polymerase III

Synthesizes new nucleotide strands by adding new nucleotides to the 3’-OH group provided by the primer (elongation)

DNA Polymerase I

Removes RNA nucleotides of the primers and replaces them with DNA nucleotides

DNA Ligase

Catalyzes the formation of a phosphodiester bond between adjacent 3’-OH and 5’-phosphate groups in a DNA molecule without adding another nucleotide to the strand

Termination

occurs when:

• Two replication forks meet

• Specific termination sequences (Ter sites) block further replication.

  • Termination protein (Tus) binds creating Tus-Tercomplex – blocks helicase

Fidelity

how accurately DNA is copied without mistakes

• proofreading

• Mismatch Repair

Proofreading

Process by which DNA polymerases remove and replace incorrectly paired nucleotides during replication

• DNA Polymerase: 3′ → 5′ exonuclease activity removes incorrectly paired nucleotides.

Mismatch Repair

Process that corrects mismatched nucleotides in DNA after replication has been completed

• Enzymes excise incorrectly paired nucleotides from the newly synthesized strand and use the original nucleotide strand as a template for replacing them

Eukaryotic DNA Replication

• The greater size of eukaryotic genomes requires that replication be initiated at multiple origins.

• Eukaryotic chromosomes are linear, whereas prokaryotic chromosomes are circular.

• The DNA template is associated with histone proteins in the form of nucleosomes, and nucleosome assembly must immediately follow DNA replication.

Aspects of Eukaryotic DNA Replication

• Origin-recgonition complex (ORC)

• Replication licensing Factor

• Eukaryotic DNA Polymerase

Origin-Recognition Complex (ORC)

Multiprotein complex that binds to an origin of replication and unwinds the DNA around it to initiate DNA replication

Replication Licensing Factor

Protein that ensures that replication takes place only once at each origin of replication; required at the origin before replication can be initiated and removed after the DNA has been replicated

DNA Polymerase

• Alpha

• Delta

• Epsilon

Alpha

Initatiation of nuclear DNA synthesis and DNA repair’ has primase activity

Delta

Lagging-strand synthesis of nuclear DNA, DNA repair, and translesion DNA synthesis

Epsilon

Leading-strand synthesis

Replication at the ends of chromosomes

• DNA synthesis at the ends of circular and linear chromosomes must differ

• ‘End-replication Problem’

• Uses:

  • Telomerase

  • G-rich 3’ Overhang

Telomerase

Ribonucleoprotein enzyme that replicates the ends (telomeres) of eukaryotic chromosomes. The RNA part of the enzyme has a template that is complementary to repeated sequences in the telomere and pairs with them, providing a template for the synthesis of additional copies of the repeats

G-rich 3’ Overhang

A guanine-rich sequence of nucleotides that protrudes beyond the complementary C-rich strand at the end of a chromosome