DNA Replication: Structure and Function

• DNA Replication Overview

Watson and Crick

• 1953: Watson and Crick's model of DNA was an example of Science Dishonesty

Rosalind Franklin

• Rosalind Franklin was the True Hero of the discovery of how DNA works
• More information about her can be found at https://www.rosalindfranklin.edu/about/facts-figures/dr-rosalind-franklin/

Nucleic Acid

• DNA and RNA are nucleic acids

Types of Ribonucleic Acids

• Deoxyguanosine monophosphate and Ribo guanosine monophosphate are examples of ribonucleic acids

DNA Structure

• DNA has a double helix structure
• Watson & Crick noted the specific pairing suggests a copying mechanism for the genetic material

Directionality of DNA

• It is important to number the carbons in DNA
• The directionality of DNA is critical, and relies on numbering the carbons

DNA Backbone

• Putting together the DNA backbone requires referring to the 3' and 5' ends of the DNA
• The last trailing carbon is also important

Anti-Parallel Strands

• Nucleotides in the DNA backbone bond from phosphate to sugar between the 3' and 5' carbons
• The DNA molecule has a "direction"
• The complementary strand runs in the opposite direction

Bonding in DNA

• Hydrogen bonds and covalent phosphodiester bonds occur in DNA
• It is important to understand how the bonds fit for copying DNA

Base Pairing in DNA

• Purines include adenine (A) and guanine (G)
• Pyrimidines include thymine (T) and cytosine (C)
• Pairing:
• A pairs with T with 2 bonds
• C pairs with G with 3 bonds

Copying DNA

• Replication of DNA involves base pairing
• Each strand serves as a template for a new strand
• New DNA is 1/2 parent template & 1/2 new DNA

DNA Replication Process

• A team of enzymes coordinates replication
• The first step of replication involves topoisomerase releasing tension
• Unwinding of DNA requires helicase:
• Helicase unwinds part of the DNA helix
• The helix gets stabilized by single-stranded binding proteins
• Building a daughter DNA strand also requires:
• Adding new complementary bases via
• DNA polymerase III

Energy of Replication

• Energy for bonding usually comes from nucleotides
• Nucleotides arrive as nucleosides
• DNA bases arrive with P-P-P, which is energy for bonding
• DNA polymerase III gets bonded

Adding Bases

• Can only add nucleotides to the 3' end of a growing DNA strand
• Must use a "starter" nucleotide to bond to
• New strand only grows from 5' to 3'

Okazaki Fragments and DNA Polymerase III

• DNA Polymerase III can only build onto the 3’ end of an existing DNA strand
• Okazaki fragments get produced
• Okazaki fragments get joined by ligase, a spot welder

Leading Strand vs Lagging Strand

• Lagging strand: Okazaki fragments get joined by ligase ("spot welder" enzyme)
• Leading strand: continuous synthesis

Starting DNA Synthesis

• Synthesis starts with RNA primers
• DNA Polymerase III can only build onto the 3' end of an existing DNA strand
• RNA primer is built by primase, and serves as the starter sequence for DNA Polymerase III

Replacing RNA Primers with DNA

• DNA polymerase I removes sections of the RNA primer
• DNA polymerase replaces the sections with DNA nucleotides
• DNA polymerase I can only build onto the 3' end of an existing DNA strand

Chromosome Erosion

• All DNA polymerases can only add to the 3' end of an existing DNA strand
• There is a loss of bases at the 5' ends in every replication
• Chromosomes get shorter with each replication
• There may be a limit to number of cell divisions

Telomeres

• Telomeres are repeating, non-coding sequences at the end of chromosomes
• The act as a protective cap
• There is a limit to ~50 cell divisions
• Telomerase is the enzyme that extends telomeres
• It can add DNA bases at the 5' end
• There are different levels of activity in different cells
• Telomerase is high in stem cells & cancers

Replication Fork Overview

• The replication fork comprises of:
• DNA polymerase I
• DNA polymerase III
• Ligase
• Okazaki fragments
• Primase
• SSB (single-stranded binding proteins)
• Helicase

DNA Polymerases

• DNA polymerase III: 1000 bases/second, DNA builder
• DNA polymerase I: 20 bases/second, editing, repair & primer removal

Editing and Proofreading DNA

• 1000 bases/second can have typos
• DNA Polymerase I
• proofreads & corrects typos
• repairs mismatched bases
• removes abnormal bases
• repairs damage throughout life
• Reduces the error rate from 1 in 10,000 to 1 in 100 million bases

Accuracy of DNA Replication

• E. coli is accurate, and copies 5 million base pairs in under 1 hour
• It divides to form 2 identical daughter cells
• The human cell is also accurate
• It copies its 6 billion bases & divide into daughter cells in a few hours
• There is only ~1 error per 100 million bases, and ~30 errors per cell cycle