Molec Cell Exam 1

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DNA Synthesis

• DNA replication: One strand of DNA serves as a template strand for the synthesis of a complementary strand in the 5’ → 3’ direction

DNA Polymerase

• Adds new necleotides to the 3’ end of the growing strand (complementary to the template strand)

DNA Helicase

• Unwinds the DNA double helix by breaking hydrogen bonds (forms a replication fork)

Single Strand Binding Proteins

• Stabilize single stranded DNA to prevent it from re-joining

Topoisomerases

• Relieves supercoiling and tension ahead of the replication fork by cutting and re-joining DNA strands

DNA Primase

• Synthesize a short RNA primer to provide a free 3’OH group required for DNA polymerase to start

DNA Ligase

• seals nicks and joins Okaizaki fragments (short length of DNA) together into a continuous strand

Directionality of DNA Synthesis

• DNA strands are Antiparallel (5’→3’, 3’→5’)

• Leading strand: synthesized continuously in the same direction that the replication fork opens

• Lagging strand: synthesized discontinuously in short segments (akazaki fragments) moving away from the advancing fork → later glued together by DNA Ligase

Replication Fork

• Y shaped DNA junction at the site where the DNA is being replicated

DNA polymerase proofreading

• DNA polymerates double checks the newly synthesized DNA using 3’→5’ exonuclease activity (removing wrong nucleotides) → DNA polymeraze then replaces it with the correct nucleotide by resuming the 5’→3’ polymerase activity

Consequences of defective proofreading

• Hypermutability: loss of exonuclease activity → severe mutator → rapid increase in number of mutation rates

• Genomic instability: uncorrected replication errors will disrupt normal gene function and cellular processes

• Disease predisposition: inherited mutation that disable proofreading can cause conditions such as Polymerase Proofreading-Associated Polyposis (PPAP) or increase risk to cancer

Replication errors

• Origin: direct mechanic and enzymatic errors by DNA polymerase during cell division

• Mechanism: the enzyme (DNA polymerase) inserts incorrect nucleotide creating insertion or deletion loops

• Fix/ Faliure: caught by proofreading or MMR (if missed it becomes permanent in daughter strand)

Spontaneous Mutations

• Origin: Intrinsic, natural chemical reactions inside the cell occurring independently of external radiation or chemicals.

• Mechanism: driven by normal cell conditions (thermal fluctuations, water attacks leading to hydrolysis)

  • Depurination: loss of a purine base

  • Deamination: loss of an amino group from a base (C→U, reading U as T)

• Fix/ Faliure: Processed by BEP (if left unfixed damaged template mispairs or halts polymerases during the following replication cycle

Mismatch Repair (MMR)

• corrects mispaired/non-complementary bases left behing by copying mistakes

  • after proofreading

  • Only occurs during and after DNA replication

Steps of MMR

1) Mismatch Detection

2) Newly synthesized strand is identified :which is the daughter strand with mistake

• in prokaryote the Parent DNA strand is methylated, daughter strand remains unmethylated

3)Recruitment and insition: MutS recruits MutL complexes

• these encode MMR proteins

4)Section of DNA is removed: Helicase and exonuclease target and remove single stranded segment with incorrect nuclease→leaves gap

5)DNA is re-synthesized: DNA polymerase fills gap DNA Ligase seals

Deamination

• loss of an amino group from a nucleotide base by a hydrolic reaction

• Causing changes such as:

  • C to U: U is read as T and will pair with A (C→G to T→A)

  • 5-Methylcytosine to T: hotspot for spontanous mutation

  • A to Hypoxanthine: is read as G and will pair with C ( A→T to G→C)

  • G to Xanthine: will pair with C, but stalls replication

Depurination

• loss of A or G bases (by spontaneous hydrolysis→ loss of glycosidic bond on base)

• leads to deletions or mutations

UV induced mutation

• a cyclobutane ring forms between adjacent T bases → T’s can not pair with A’s

• leads to distorted strand

• Repaired by NER/photolyase

Base Excision Repair (BER)

• General repair mechanism for nucleotide damage (chemically altered or modified single base)

  • damaged base is replaced before the next replication cycle

  • Operates throughout the entire cell cycle

BER steps

1) Recognition and removal of damaged base: DNA glycosylase identifies and removes the base

2) Addition of new repaired base by DNA polymerase

3) DNA Ligase seals the nick in the backbone

Nucleotide Excision Repair (NER)

• fixes thymine dimers and multiple base damages (Bulky damages)

  • Defects can lead to Xeroderma Pigmentation

Transposons

• segments of DNA (motile) that can change its location inside the genome

  • LINEs: Long Interspersed Nuclear Elements (~6000-8000BP), Autonomous (move independently)= reverse transcriptase and endonuclease

  • SINEs: Short Intersperesed Nuclear Elements (~100-400BP), Non-autonomous (Needs a partner LINE to move) = non coding sequences

Bacterial Transposons

• May carry antibiotic resistance genes

• Occures by two pathways

  • Replicative transposons: copy-paste

  • Non-replicative transposons: cut-paste

Replicative transposons

• the enzyme transposase cleaves the insertion sequence at the end of the inverted repeat of the donor site

• cleaves a random target site

• insertion sequence: cu from donor site pasted in target site

• result: insertion sequence moves from one site in the genome to another

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Replicative Transposon

• a copy of the insertion sequence is made by local DNA replication and pasted into target site

• result: copy of insertion sequence stays intact and appears in target site

Retrotransposition

• done by reverse transcriptase synthesizing a DNA copy of transcribed retrotransposon

• Only in eukaryotes

• can generate many copies of original mobile element

• in some plants retrosposons account for most of the DNA in the genome

• reshape genome structure, alter gene expression, and contribute to genetic diversity

DNA Virus infection cycle

1) Enter host cell (nucleus)

2) Replicate viral genome

3) Protein synthesis (transcription, translation)

4)Assemble progeny

5) Release

RNA virus infection cycle

1) enter host cell (cytoplasm)

2) synthesize proteins (translation)

3)Assemble RNA replicase complex

4) Synthesize complementary RNA

5) goes two ways:

Retroviruses

Use reverse transcriptase

Organization of Eukaryotic chromosomes

DNA double bonded helix→ Nucleosomes→ Chromatin fiber → higher order loops and topological domains → condensed metaphase chromosomes

Nucleosomes

• DNA wrapped around 8 Histones (repeating unit of chromatin)

Higher order chromatin structures

• compact meters of DNA into a microscopic nucleus while precisely controlling gene activity, DNA repair, and replication.

Histone and chromatin associated proteins

• organize long DNA molecules into compact structures inside the cell nucleus while controlling gene access

The four core histone proteins

• H2A, H2B, H3, and H4

• They bind together to form a protein disc called a histone octamer

role of Histone H1

• It acts as a linker histone

• It binds the entry/exit sites of DNA on the nucleosome (locks the strand into place to help fold the 10-nm fiber into a more compact 30-nm chromatin fiber)

chromatin-associated remodelers

• use ATP energy to slide, evict, or modify nucleosomes

  • This shifts DNA packaging to dynamically open or close specific areas of the genome

Euchromatin

• Loosely packed

• Accessible

• Transcriptionally active (genes are on).

• Gene rich

Heterochromatin

• Highly condensed

• inactive/ Silenced (genes are off)

• inaccessible

• Repeat Rich

post-translational modifications (PTMs)

• use:

  • Phosphorylation: kinases add phosphate groups to amino acids creating a physical block or an electrical shift that activates or halts an enzyme

  • Cleavage: targeted cutting triggers a sudden release of biological activity in proteins (by insuline protein)

• Alters gene access

  • chemical groups added to histone tails (acetylation/methylation) change the physical tightness of the DNA-histone grip and serve as binding docking sites for gene regulatory proteins

three essential functional elements required for eukaryotic chromosome maintenance and inheritance

• Centromeres, telomeres, and origins of replication

structural hallmark of a eukaryotic centromere

• Repetitive DNA packed with specialized CENP-A histone variants instead of standard H3, which epigenetically marks the region for chromosome segregation.

primary role of the centromere during cell division

• consists of repetitive G-rich sequences (like TTAGGG) that form a single-stranded overhang

  • this overhang loops back into a T-loop stabilized by the shelterin protein complex

telomeres solve

• end-replication problem

• act as non-coding buffer zone (prevents loss of genetic data during shortening of DNA during replication)

Eukaryotic chromosome origins of replication

• enriched with A and T bases

• A-T base pairs share only two hydrogen bonds (compared to three in G-C pairs), making it energetically easier for replication machinery to pull the strands apart

role of the Origin Recognition Complex (ORC)

• binds directly to origins of replication during the cell cycle to license and initiate bidirectional DNA duplication

Chromatin condensation

• tightly packs DNA into Hetorochromatin (physical tightening of DNA)

• Causes gene innactivation

Histone modification

• chemical tags on Histone Tails

DNA Methylation

• Chemical tags directly on DNA bases

• Adding methyl groups to specific amino acids (like H3K9 or H3K27) recruits silencing proteins

• Methyl groups (usually added to cytosine bases in CpG islands) physically block transcription factors from binding to the promoter region

• Silencing

Histone Deacetylation

• Removing acetyl groups increases the positive charge of histones, making them bind DNA more tightly

How do DNA methylation and histone modifications work together to lock a gene silent

• Methylated DNA attracts Methyl-CpG-binding domain proteins (MBDs)

• These proteins recruit Histone Deacetylases (HDACs), which strip acetyl groups and force the chromatin to condense

Xist (X-inactive specific transcript)

• makes a long non-coding RNA molecule (lncRNA) that physically coats the X chromosome from which it is transcribed, triggering silence

X-chromosome inactivation

• Female mammals shut down one of their two X chromosomes to match the single X chromosome dose found in males

Epigenetic inheritance

• The chromosome which is silenced is tagged (the tags are part of epigenetics)

• DNA Methylation : cells add methyl groups to DNA (small chemical tags) to insure inactivity

• Histone modifications: histones receive inhibitory marks, and active histones are removed

• Miotic Memory: daughter cells of inactive chromosome will have tags to ensure inactivation

Promoters

• Act as the primary binding site for RNA polymerase and general transcription factors to initiate transcription

• Found just upstream (at the 5' end) of the main coding region

• Includes:

  • core promoter: positions the enzyme correctly (often containing a TATA box)

  • proximal promoter: help modulate the basal rate of transcription

Regulatory sequences

• Controls when, where, and at what level a gene is expressed

• upstream, downstream, or inside introns

• Includes:

  • enhancers: boost transcription when specific activator proteins bind

  • silencers: repress transcription when repressor proteins bind

Exons

• sequences retained in the mature messenger RNA (mRNA) after splicing

• expressed/ kept sequences

• Segments interspersed throughout the transcribed portion of the gene

• many exons contain the protein-coding sequence (CDS) translated into amino acids

• contain the untranslated regions at the ends of the transcript

Introns

• Transcribed into initial pre-mRNA but completely removed during RNA splicing before the mRNA is translated

• Found in intervening non-coding sequences that alternate with exons in the DNA and pre-mRNA

• Not expressed/ cut out

• Contain vital signals like splice donor and acceptor sites, and sometimes harbor auxiliary regulatory motifs or alternative splice variants

Unregulated regions (UTR’s)

• Regulate translation efficiency, mRNA stability, and subcellular localization, though they are not translated into protein

• How and when gene expression happens

• Found at both ends of the processed mRNA transcript, originating from terminal exons

• Include:

  • 5′ UTR: Sits before the start codon, assists in ribosome binding and translation start

  • 3′ UTR: Sits after the stop codon, contains polyadenylation signals and binding sites for microRNAs that control mRNA degradation

Genome size and density comparison

Humans have:

• Large introns

• Extensive repetitive DNA

• Regulatory sequences spread over large regions

Tandem repeats

• repeats adjacent to each other (together)

• found in centromeres and telomeres

• simple repeats

Transposable elements (interspersed repeats)

• can move or copy themselves

• make up a large fraction of eukaryotic genome

• include:

  • retrosposons (copy and paste, LINEs, SINEs): class I

  • DNA transposons (cut and paste): class II

Repeated sequences

• influence chromosome structure

• Drive genome evolution

• Affect gene regulation and stability

Polypeptides

• linear polymer of amino acids

• synthesized by ribosomes

• Opposes directionality:

  • synthesis occurs from N terminus to C terminus

• Connected by covalent peptide bonds

Polypeptide synthesis

• Condensation/ Dehydration reaction: two amino acids are joined by removing a water molecule

A protein’s amino acid sequence

• consists of:

  • Primary structure: amino acid sequence (bases) (covalent peptide bonds)

  • Secondary structure: alpha-helices and beta sheets (hydrogen bonds)

  • Tertiary Structure: 3D fold driven by side chain interactions (side chains)

  • Quartery structure: assembly of multiple peptide subunits (hydrogen bonds, ionic bonds, hydrophobic interactions, and occasionally disulfide bonds or covalent links)

Molecular chaperone

• help protein fold correctly

Misfolding consequences

• aggregation: loss of function or toxic gain of function

• linked to neurodegenerative disease

Protein Folding

• spontaneous process where polypeptide adopts its native energetically favorable confirmation

protein misfolding consequences

• disrupts proper three-dimensional conformation

• lead to a loss of function or gain of toxic properties ( linked to disease)

Prion disease

• Infectious misfolded proteins template the conversion of healthy proteins into a toxic, aggregated state via a self-propagating cascade

• touches normal protein and acts like a mold or template to unfold it

• can lead to Transmissible spongiform encephalopathies (TSE): demaged brain tissue

Disulfide bonds

• form between thiol groups of cystein residues

• Stabilized tertiary + quarternary structures of proteins

• Occurs in Oxidizing/ reducing environments ( ER, Cytoplasm)

Denaturation

• loss of 3D structure due to disruption of non-covalent bonds by heat, pH extremes, or chemicals

• can be reversible

• Bad environment→ protein loses shape→protein loses function

• Breaks 2-4 structure of proteins

Reducing Agents

• reduce/ break covalent disulfide bonds, convertthem into free thiols (beta-mercaptoethanol)

• breaks 3-4 structure of proteins

Ubiquination

• Ubiquitin cahin is attached to tag a protein for degradation

• polyubiquitin chains targett proteins

• proteosome degrades ubiquitilated protein into peptides (ATP dependent)

Elastin fibers

• a rubberlike elastic meshwork present in the extracellular matrix of some cell types

• allow tissues such as skin, arteries and lungs to stretch and recoil without tearing

Fibrillar collagens

• major structural proteins of connective tissues

• built of triple helices of procollagen polypeptides

Macromolecules

• Large, complex molecules vital for life, built from smaller building blocks

Monomers

• single subunit of polymers

Dehydration/ condensation synthesis

• building polymers by covalently bonding monomers

• releases a water molecule

Hydrolysis

• breaking down polymers by adding a water molecule

four major classes of macromolecules

1) Carbohydrates

2) Lipids

3)Proteins

4)Nucleic Acids

Carbohydrates

• subunit is monosaccharides (simple sugars)

• are carbon rings or chains

• provide short term energy and support

Lipids

• glycerol + fatty acids

• For long term energy (triglycerine), creating cell membrane (phospholipids), and structural support (steroids→cell signaling)

• include:

  • Saturated fatty acids: no double bond, pack tightly

  • Unsaturated fatty acids: double bonds, create kinks

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Proteins

• monomers are amino acids

• made from amino, carboxyl, and r-groups

• provide enzymes, structure, transport and defense

• linked by peptide bonds

Nucleic acids

• monomers are nucleotides

• made from phosphate, pentose sugan, and nitrogen base

  • store and transmit genetic info

Chemical Interactions

• covalent bonds

• ionic bonds

• hydrogen bonds

• van der waals interactions

Sickle Cell Disease

• Changing a single amino acid in hemoglobin alters its structural folding

• causes the proteins to clump into rigid fibers, distorting red blood cells into a sickle shape and reducing oxygen transport