Lecture 8 - Chromatin and Nuclear Structure Flashcards
What is supercoiling?
Stressed state of DNA - more/less than 10.4 bp per helical turn - this is known as supercoiling. Negatively supercoiled = less, positively supercoiled = more
What are topoisomerases?
Positive supercoils accumulate ahead of replication or transcription complexes
Effect is transferred until a domain boundary/anchor point is reached
Topoisomerases relieve the stress
Topoisomerases sense supercoiling and respond
Type 1 cuts one strand, passes the other through then ligates it
Type 2 cuts both strands then passes an unbroken double strand through
What are condensins?
Condensin one of the main components of SMC complex - functions as a motor protein to extrude DNA loops
Move DNA relative to itself in vivo in the cell. Condensins are fixed and it’s DNA that’s moving through their locations in the nucleus
What is the difference between heterochromatin and euchromatin?
Heterochromatin Highly compacted Generally inactive Gene poor Often situated at the periphery of the nucleus Usually replicated late in S phase
Euchromatin Less compacted Gene rich Contains active genes Usually replicated early in S phase
Describe how the DNA damage response can be visualised experimentally
Experiment:
UV light creates a stripe of damage.
H2AX is phosphorylated locally
Phosphorylated H2AX helps attract repair factors
Describe how epigenetic information is copied
Daughter molecules tend to receive about half of the parental histones - we have molecules that can bind the ones that are received and recapitulate the modifications in the others. These are so called reader writer complexes and they ensure that daughter molecules get the same pattern
Describe how DNA methylation takes place and is copied
DNA methylation is added by specific enzymes specifically added at CG dinucleotides
Methylation is reversible
Some cells have it some don’t - this determines whether a cell can express a particular gene
Regulates gene activity at the level of binding of transcription factors
CpGs have to be copied into daughter cells
During development there is the addition of methylation at CpG islands in our promoters. Upon replication to create daughter cells only half will go into daughter molecules but because this is effectively a palindromic sequence there is recapitulation of the original pattern - enzymes that recognise the phosphorylated CGs and add it to the other strand in the daughter molecule
Describe nuclear organisation of DNA
Spatial organisation of chromosomes in the nucleus - organised in very specific way.
Whole chromosomes occupy distinct territories
The statistical likelihood of two sequences being close to each other can be determined which translates to whether or not they exist in the same protein complex
Chromosomes have preferred locations relative to others, and their position is heritable
Genes on different chromosomes can be part of the same megacomplex and regulated together
Sequence proximity can be measured
Describe how the proximity of two sequences can be measured
Proximity ligation method:
An intact nucleus is taken and a chemical agent applied that will crosslink bits of template with particular protein factories. You then fragment the whole genome such that those bits that were in proximity to one another are anchored together by those crosslinks. This particular version of the method then adds a flag to the tails/loose ends and an enzymatic ligase will join those two ends. There is then another fragmentation step such that you break up all the bits that were bound to this and the flagged bits are isolated and then sequenced. This can be done on a whole genome level and the frequency of one sequence being aligned with another can be determined - frequency map generated for a nucleus which can be spatially mapped onto genome. Some regions within chromosomes are frequently associated with one another - topologically associated domains. Maps frequency of interaction - this is not random
