Molecular Biology: DNA Replication

DNA Strands

• DNA strands exhibit an antiparallel arrangement
• Nucleotides are added by DNA polymerases exclusively to the 3' end of a growing strand

DNA Replication

• Leading strands replicate toward the replication fork
• Lagging strands replicate away from the replication fork
• Leading strand: synthesized as one continuous piece within the replication "bubble"
• Lagging strand: consists of 100-200 nucleotide Okazaki fragments
• Okazaki fragments are joined by DNA ligase
• DNA polymerases are unable to initiate DNA synthesis

Initiation of Replication

• A primer initiates a new DNA chain, composed of RNA and around 10 base pairs long
• Primase: an enzyme that initiates replication by joining RNA nucleotides, thus creating a primer
• Leading strand replication requires one primer, whereas each Okazaki fragment requires a primer
• Primers are replaced by DNA using another DNA polymerase, then DNA ligase joins the fragments

Overview of DNA Replication

• DNA strands have a leading and lagging strand at the replication "bubble's" opposite ends

Enzymes in DNA Replication

• Helicases: untwist and separate the DNA helix
• Single-strand binding proteins: keep separated strands apart

DNA Repair

• Over 100 enzymes are involved in DNA proofreading and repair
• DNA nucleotides are proofread by DNA polymerase during synthesis
• Mismatch repair: corrects incorrectly paired nucleotides using enzymes
• Nuclease enzymes: cut DNA

Nucleotide Excision Repair

• Damaged DNA sections are excised
• A polymerase and ligase fill the excised section

The 5' Problem

• DNA polymerase adds nucleotides to the 3' end, thus there is no way to complete the 5' end
• Prokaryotes with circular DNA avoid this issue
• Eukaryotes have telomeres

Telomeres

• Consist of 100-1000 repeated short DNA sequences, like TTAGGG in humans

Telomerase

• An enzyme contains RNA, further extends the 3' end for 5' end completion

Telomerase and Cell Division

• Telomerase is present only in germ-cell lines
• Telomeres in somatic cells shorten with division
• Telomere shortening may limit lifespan

Telomere Loss

• A faster rate of telomere shortening correlates to a shorter lifespan in birds

How Telomeres Contribute to Ageing

• Telomerase is a reverse transcriptase that restores telomere ends
• Telomerase is not expressed in human somatic cells
• Cell machinery cannot replicate to the chromosome's end
• Telomeres get shorter every time the cell divides
• Cells stop dividing when telomeres become critically shortened
• Cellular senescence occurs
• Ageing may be caused by the accumulation of senescent cells

Telomeropathies

• A shortening disorder, occurs due to defects in telomere maintenance machinery

Comparative Genomics

• Comparative genomics allows scientists to understand genome functions, and how the genome has evolved
• Bat telomeres do not shorten with age in the species with the longest lifespan

Genetics Section Objectives

• Meiosis, mitosis, and inheritance should now be understood
• Comprehension of Mendel's work in genetics
• Grasp of the chromosomal inheritance basis
• Understanding of DNA structure, replication, and repair
• RNA transcription
• Understanding of protein translation
• Genes on DNA code for RNA, which produces proteins that perform functions in cells

Ribosomes

• Ribosomes have a 3-D representation with tRNA

One Gene - One Enzyme Hypothesis

• Bread mold Neurospora crassa: can grow with just agar, salts, glucose, and biotin
• X-rays were used to cause mutations, preventing the strains from growing with just those nutrients
• Scientists supplemented the agar to discover required amino acids and nutrients for growth

One Gene - One Polypeptide

• Many proteins have several polypeptide chains coded by a single gene
• It is acceptable to refer to gene products as proteins rather than as polypeptides

Linking Genetic Code to Polypeptides

• The DNA genetic code consists of the letters A, C, G, and T, in a linear array
• RNA acts as an intermediate "language"
• Linear arrays of amino acids then encode protein "language"

Translation Differences

• Prokaryotes lack a nucleus
• Messenger RNA is immediately translated into polypeptides
• Eukaryotes transcribe RNA, which is then processed into mRNA

Triple Code

• 3 DNA bases code for a single amino acid

Template Strand

• Coded DNA strand for a gene

Codon

• The mRNA triple code for an amino acid
• Codons are read in the 5’ to 3’ direction

Reading Frame

• Triplet grouping

Codons

• Polypeptides usually start with methionine
• There is built in redundancy, however there is no ambiguity
• Codons are read in triplets from 5' to 3'
• The DNA triplet codes for "start" and "stop"

Universal Code

• DNA's code is nearly universal
• Codon CCG codes for proline in everything
• sections of DNA transferred from one organism to another produce meaningful protein

RNA Polymerase

• RNA polymerase pries apart the DNA helix and hooks together the RNA nucleotides
• RNA polymerase can only add nucleotides at the 3' end
• A promoter region of DNA is where RNA polymerases attach and initiates transcription

Transcription Unit

• A transcription unit is a stretch of transcribed DNA
• A terminator is a DNA sequence that signals the end of transcription

Post-Transcription RNA Modification

• The 5' cap modification is a modified guanine nucleotide that protects mRNA from enzymes and helps attach it to ribosomes
• The poly(A) tail: consists of 50-250 adenine nucleotides, inhibits degradation, helps ribosomes attach, and helps mRNA leave the nucleus

Introns

• Intron excision involves RNA acting as an enzyme

snRNPs

• Small nuclear ribonucleoproteins are found in the nucleus and lead to spliceosome formation, they catalyze excision of introns

Spliceosome

• Several snRNPs and proteins splice the exons into mRNA, about as big as a ribosome

Alternative RNA Splicing

• Intron presence can control activity of genes
• Discrete structural or functional regions of proteins are generally coded by an exon
• Introns allow crossing over in non-coding regions

Exons and Introns

• Exons consist of sections of DNA and RNA that become ‘expressed’ or translated into protein
• Introns consist of non-coding, intervening regions of RNA and DNA
• Both introns and exons are transcribed into preRNA

mRNA

• mRNA represents the coded message for proteins

tRNA

• tRNA transfers amino acids from the cytoplasm into the ribosome
• Each TRNA is specific to one or several codons and coded as an anti-codon

Ribosomes

• Ribosomes are composed of rRNA and proteins, in a large and small unit
• Act to couple tRNA and mRNA
• Has three binding sites for tRNA

Polypeptide Initiation

• Small RS binds to mRNA at 5' cap and an initiator tRNA
• Initiating factors are used
• GTP is the energy source

Signal Peptides

• Indicates the destination of a protein at leading end of a polypeptide

SRP (SRP RNA)

• Signal-recognition particle are proteins that recognize signal peptides to move them to specific locations
• Only found in Eukaryotes

Eukaryotes vs Prokaryotes

• In prokaryotes, transcription and translation of the same mRNA can occur simultaneously
• Prevents additional regulation of cell activity as nucleus prevents simultaneous transcription and translation

Point Mutations

• Changes in one base pair of a gene which is passed to offspring in gametes or reproductive cells
• A base-pair substitution can lead to one of the main point mutations

Substitution Mutation

• Involves one nucleotide AND its partner with another nucleotide which is replaced

Silent Mutation

• Exhibits no changes or affects

Missense Mutation

• Coding protein changes to another non-stop codon and sometimes is good, sometimes bad

Nonsense Mutation

• Coding changes to a stop codon prematurely terminates a polypeptide and almost always bad

Insertions/Deletions

• Addition or missing a nucleotide pair in a gene which results in a frameshift mutation, and wrong amino acids being added

Comparative Genomics

• Comparative genomics enables an understanding of how our own functions and how we evolved genomes.
• Telomeres do not shorten with age in longest life bats.