DNA Replication and Repair

The Replication Fork

• At a replication fork a newly synthesized strand is made on a template strand running 5' to 3'
• Another strand is synthesized on a template strand running 3' to 5' direction
• This makes the replication fork asymmetrical

DNA Polymerase 3

• DNA polymerase 3 always builds a new strand in the 5' to 3' direction
• DNA polymerase 3 reads the template in the 3' to 5' direction, but writes in the 5' to 3' direction

Leading and Lagging Strands

• The newly synthesized DNA strand made continuously in the 5' to 3' direction toward the replication fork is the Leading Strand
• The template strand guides the synthesis of the leading strand and runs in the 3' to 5' direction
• All newly synthesized strands are either leading or lagging strands depending on their direction relative to the replication fork
• When the synthesized DNA approaches the nearest fork, they are either leading or lagging

DNA Polymerase as a Self-Correcting Enzyme

• DNA polymerase cleaves an incorrectly added nucleotide from the growing strand
• It replaces an incorrectly added nucleotide with the correct nucleotide
• DNA Polymerase 3 adds incoming nucleotides to the 3' end of the newly synthesized strand

Proofreading

• In polymerizing mode (P), DNA polymerase synthesizes DNA, and in proofreading mode (E), it can correct errors
• Catalytic sites for polymerization (P) and editing (E).
• Incorrect nucleotide addition causes the newly synthesized strand to temporarily unpair.
• The 3' end of the strand moves to the editing site (E) for removal of the incorrect nucleotide.

Steps in Proofreading

• DNA polymerization must proceed in the 5' to 3' direction for proofreading
• High-energy bonds must be available to continue DNA replication.
• Hydrolysis of incoming deoxyribonucleoside triphosphate provides energy for polymerization.
• Hydrolysis of phosphate bond at 5' end of growing strand provides energy for polymerization
• DNA polymerase cleaves an incorrectly added nucleotide before adding the next one

Short Lengths of RNA Act as Primers

• Short RNA sequences, known as primers, are essential for DNA replication
• These RNA primers provide a starting point for DNA polymerase to begin adding nucleotides
• Primers are synthesized by primase during DNA replication
• RNA primers are complementary to the single-stranded DNA template

Enzymes Required to Synthesize Lagging Strand

• RNA primers of about 10 nucleotides are extended by DNA polymerase III to form Okazaki fragments of approximately 1200 nucleotides
• RNA primers are removed by nucleases that degrade the RNA strand in RNA-DNA hybrids
• Gaps left by primer removal are filled by DNA polymerase I, which can proofread as it works
• DNA ligase joins DNA fragments by forming phosphodiester bonds between fragments

DNA Ligase

• DNA ligase joins together Okazaki fragments on the lagging strand during DNA synthesis via ATP hydrolysis
• ATP and GTP are commonly used as energy currencies in cells

Telomeres

• Telomerase replicates the ends of eukaryotic chromosomes

DNA Replication Risks

• Replication starts at origins and continues to chromosome ends
• The leading strand is fully synthesized
• The lagging strand cannot always be completely synthesized
• DNA replication may result in the loss of chromosome ends

Mechanism for Chromosome End Protection

• Telomerase extends the template strand beyond the DNA to be copied
• The extended template allows primase to lay down RNA primers
• Telomerase uses RNA as a template to synthesize telomere DNA
• After replication, the lagging strand is lengthened

Telomere Lengths

• Telomere length varies by cell type and with age
• Telomeres are repetitive DNA sequences at the ends of chromosomes, protecting them from damage and fusion
• Stem Cells and Germ Cells maintain longer telomeres to divide extensively over a lifetime
• Somatic cells generally have shorter telomeres because they divide fewer times, losing a small portion of the telomere with each division

Types of DNA Repair

• DNA damage occurs continually in cells
• Cells have various mechanisms for repairing DNA
• A DNA mismatch repair system removes replication errors that escape proofreading
• Double-strand DNA breaks require a different strategy for repair
• Homologous recombination can flawlessly repair DNA double-strand breaks
• Failure to repair DNA damage can have consequences for a cell or organism
• A record of the fidelity of DNA replication and repair is preserved in genome sequences

DNA Damage

• DNA damage occurs continually in cells

Chemical Reactions That Damage DNA

• Depurination and deamination are the most frequent chemical reactions that create serious DNA damage in cells
• Depurination removes guanine or adenine, leading to potential mutations
• Deamination converts cytosine to uracil

Impact of DNA Damage

• Both depurination and deamination can alter the genetic code without breaking the DNA's phosphodiester backbone
• Modifications can lead to errors during DNA replication and repair, contributing to mutations
• Common sources of DNA damage don't disrupt the structural integrity of the DNA helix

External Factors That Damage DNA

• Chemical changes can occur spontaneously, triggered by environmental factors, reactive molecules, and cellular processes

UV Radiation

• Ultraviolet radiation in sunlight can cause the formation of thymine dimers

Consequences of unrepaired DNA modification

• If left unrepaired, chemical modifications of nucleotides produce permanent mutations

Error Rates

• DNA replication without proofreading has an error rate of one mistake per 10^5 nucleotides copied
• With proofreading but without mismatch repair, the error rate is one mistake per 10^7 nucleotides copied
• Adding mismatch repair to proofreading improves the error rate to one mistake per 10^9 nucleotides copied

Errors in DNA replication

• Errors made during DNA replication must be corrected to avoid mutations
• Mismatch repair eliminates replication errors and restores the original DNA sequence

Repairing Double-Strand DNA Breaks

• Cells can repair double-strand breaks in one of two ways: nonhomologous end joining and homologous recombination

Homologous Recombination

• Homologous recombination can repair double-strand breaks
• Invading strand is released and complementary base-pairing allows broken helix to re-form. DNA synthesis continues using complementary strands from damaged DNA as a template
• DNA ligation results in a accurately repaired double-strand breaks

Consequences of DNA Repair Failure

• Failure to repair DNA damage can have consequences for a cell or organism
• A single nucleotide change can cause diseases like sickle cell anemia

Explore DNA replication fidelity, repair mechanisms, and the consequences of errors. Understand the roles of key enzymes like DNA polymerase III, and the impact of damage like depurination and deamination on genome stability. Investigate the impact of replication fork asymmetry.