Bacterial chromosomes have a single origin, termed ori, whereas eukaryotic organisms can have many not sequence specific.

Can broadly split the process into three parts. Initiation, elongation and termination

Primase and DNA polymerase A work in close association.

This is the S-phase CDK.

• Replication origin licensing.
• All of this occurs in G1 phase

Orc1-5 complex.

Cdc6 is only present in G1 phase.

DNA replication pathway in humans. Orc complex binds the origin of replication.

Workflow 1. Cross-linking 2. Chromatin fragmentation 3. Immunoprecipitation of chromatin 4. DNA recovery and purification 5. Sequencing of DNA

ChIP-seq is concerned with testing for protein DNA interactions.

NER * Helix distortion * ERCC6 and 8 mutation -- Cockayne's syndrome * XP proteins (XPE or DDB2, XPC, XPA) mutated in xeroderma pigmentosum. * TFIIH, the same helicases as seen in DNA replication.

BER * For non-helix distorting base lesions. * Base is not present, a gap is present in DNA. * Specific DNA glycosylases used for identification. * APEX1 and APEX2 (AP endonucleases): responsible for end processing * An AP site (apurinic/apyrimidinic site). * The exposed 3' OH is available to a replicative polymerase. * Ligation performed by ligase * Short or long patch BER is possible.

In HR, * MRN -- begginings of dsDNA resection. * The PARP1 protein is active on ssDNA. * Free 3' ends made available, crucial for later DNA pol binding. * RPA binds, coats, the ssDNA.<br /> * Rad51 searches for strand for invasion. * Strand invasion carried out by sister chromatid, etc. * D-loop formation.

May have. * DSBR * SDSA * BIR

NHEJ relies on microhomologies. Doesn't require homologous sequence from another source. * Ku protein instrumental in identification of DSB and recruits DNA-PKcs. * DNa-PKcs autophosphorylates. * DNA ends processed by Artemis. * LIG4 and XCRR4 are needed for strand ligation.

NHEJ and HR can be compared.

TEs are transposable elements.

Transposons are mobile DNA elements.Can move throughout the genome. Can be catagorised as class 1 (retrotransposons) or class 2 (DNA transposons).

Class 1 comprises TEs with LTRs, retroposons (LINE), SINEs.

Class 2 comprises TEs that operate under replicative transposition or non-replicative transposition. Replicative transposition (nick and paste) -- a total of two TEs as an end result, one as part of the donor and one as part of the target sequence. cointegrate.

Non-replicative transposition (cut and paste) - only one TE generated, in the target.

Examples of DNA-only transposon:

The initial sample consisted of 1 band. F = 0. 1st generation = The sample overall less dense, still one band. Intermediate density. dsDNA made of one strand heavy and one light. After 2 generations, there was a band for intermediate density and for strands of just light, N-14, dsDNA.

Three methods of replication were initially hypothesized: * Conservative * Semi-conservative * Dispersive

Semi-conservative method was eventually determined to be correct based on empirical evidence from Messelson & Stahl, in 1958.

Parental strands consist of N-15 isotopes. Replicated daughter strands consist of N-14 isotopes. The CsCl ultracentrifugation process creates density gradient. This allows DNA fragments of different densities to migrate and form a band at the point at which their buoyant density equals that of the salt.

1. E.coli grown in an heavy nitrogen, N-15, enriched medium
2. Then transferred to N-14 based media to reproduce
3. As several points in the experiment, cells were lysed and underwent ultracentrifugation through a CsCl concentration gradient

• Denature
• Annealing primer
• Synthesize new DNA with polymerase

• High speed
• Extreme sensitivity