Researchers have succeeded in depleting AND-1, a key protein for DNA replication, by using a recently developed conditional protein degradation system. Consequently, they were able to gain unprecedented access to the mechanism behind how AND-1 works during DNA replication and cell proliferation in vertebrate cells, demonstrating that AND-1 has two different functions during DNA replication mediated by different domains of AND-1.
DNA is often referred to as the "blueprint of life"; in order for living organisms to function, it is vital that all cells share the same blueprint. This is made possible by the process of DNA replication, where the DNA is accurately copied and distributed before the cell multiplies. Replication underpins all biological inheritance, and is supported by a whole range of biochemical pathways designed to ensure that it occurs without error and at the right speed. Failure to do so may have catastrophic consequences, including cancer: understanding the specific mechanisms behind this highly complex procedure is of the utmost importance.
The AND-1/Ctf4 protein is a key player in DNA replication, and is found in a vast range of living organisms, from fungi to vertebrates. Ctf4/AND-1 is essential in some organisms, but whether it is an essential gene for cell proliferation in vertebrates has not been shown experimentally. Moreover, how loss of AND-1 affects cell proliferation is not known.
In order to address this question, the team combined the use of two unique systems, the DT40 cell, a type of avian cell that is particularly suited to genetic engineering, and the auxin-inducible degron (AID) system, a means to realize selective depletion of a target protein. With these, they successfully established the and-1-aid cell line, in which a modified version of the AND-1 protein is degraded in a few hours after adding auxin, a type of plant hormone. This cell line enabled them to analyze the acute consequence of AND-1 loss, giving unprecedented insight into the role it played.
When done correctly, DNA replication should result in the formation of new double-stranded DNA helices. Authors used transmission electron microscopy (TEM) to visualize DNA replication intermediates and observed newly synthesized DNA having abnormally long single stranded DNA at the fork branching point in the absence of AND-1. They hypothesized that this was due to a DNA cleaving enzyme, a nuclease, disrupting the process of strands being disassembled. On further addition of a compound that suppresses the action of a particular nuclease, MRE11, they were able to successfully revert the abnormal replication fork phenotype and recover cell division, explicitly demonstrating the key role played by AND-1 in preventing nascent DNA cleavage by the nuclease during replication. Further analysis revealed that a specific part of the protein called WD40 repeats was responsible for preventing the accumulation of damage to the strand.
Further to these breakthrough findings, the study highlights the successful combination of cutting-edge techniques to realize conditional inactivation of specific proteins; the new cells were in fact developed over a single month. This leaves the exciting prospect of the method being applied to study other genes and processes which are otherwise difficult to target, leading to new insights into how cells work.
https://www.nature.com/articles/s41467-018-05586-7
Latest News
Which of the two DNA strand…
By newseditor
Posted 16 Jun
Microglia depletion prevent…
By newseditor
Posted 16 Jun
Colorectal cancer stem cell…
By newseditor
Posted 16 Jun
Paranoia in the brain
By newseditor
Posted 16 Jun
In-vitro 3D culture of func…
By newseditor
Posted 15 Jun
Other Top Stories
Why only some people are easily addicted to drugs?
Read more
Why some individuals seem to be more susceptible to PTSD
Read more
Link between the gut and death by sleep deprivation
Read more
Childhood trauma affects the timing of motherhood
Read more
Elevated love hormone levels and higher satisfaction with life in y…
Read more
Protocols
Bioengineered human colon o…
By newseditor
Posted 14 Jun
Development of an efficient…
By newseditor
Posted 12 Jun
A co-culture system of macr…
By newseditor
Posted 10 Jun
Analysis of 3D pathology sa…
By newseditor
Posted 08 Jun
Long-term expandable mouse…
By newseditor
Posted 07 Jun
Publications
Pathways for macrophage upt…
By newseditor
Posted 16 Jun
Common and distinct neural…
By newseditor
Posted 16 Jun
Strand-resolved mutagenicit…
By newseditor
Posted 16 Jun
Innate-like T cells in live…
By newseditor
Posted 16 Jun
Membrane to cortex attachme…
By newseditor
Posted 16 Jun
Presentations
Myelin plasticity in the ve…
By newseditor
Posted 10 Jun
Hydrogels in Drug Delivery
By newseditor
Posted 12 Apr
Lipids
By newseditor
Posted 31 Dec
Cell biology of carbohydrat…
By newseditor
Posted 29 Nov
RNA interference (RNAi)
By newseditor
Posted 23 Oct
Posters
A chemical biology/modular…
By newseditor
Posted 22 Aug
Single-molecule covalent ma…
By newseditor
Posted 04 Jul
ASCO-2020-HEALTH SERVICES R…
By newseditor
Posted 23 Mar
ASCO-2020-HEAD AND NECK CANCER
By newseditor
Posted 23 Mar
ASCO-2020-GENITOURINARY CAN…
By newseditor
Posted 23 Mar