For every rule in biology there is an exception. This causes problems in teaching. Do you give the rule and leave out the exception, effectively lying to the people you are teaching or do you give them the rule and exception and making rule mean much less? This is a real problem. Well I have just discovered eukaryotes have broken a golden. They have operons. I was always taught eukaryotes don’t have operons, only prokaryotes do.

For anyone needing to be reminded what an operon is it is several genes all on the same mRNA molecule. One name for this is polycistronic mRNA (cistron is an old name for what we know as a protein coding gene). Several genes have a single promoter and a terminator of transcription controlling them all. On this polycistronic mRNA are several Shine-Dalgarno sequences (bacterial ribosome binding sites) so a ribosome binds in front of each gene and translates it until it reaches a stop codon and falls off. The classic example is the lac operon. It makes 3 proteins used by E. coli to break down the sugar lactose. The reason why you would want a single promoter to control the expression of several proteins is because if they have related function like in the lac operon you want them all to be turned on at the same time and at the same level of expression therefore making the same number of proteins.

Classically eukaryote have monocistronic mRNA (one gene per mRNA) but their is more to this story. Strange transcripts have been found in animals from the simple nematodes to complex mammals such as us. One type of mRNA that has been called operons by some people in C. elegans uses something called trans-splicing. A gene is expressed with one promoter but contains 2 protein coding regions. But this does not mature into a polycistronic mRNA but during RNA processing the introns are removed and the two coding regions are separated. But how does the downstream RNA resist degradation by RNases? It has a SL-exon from another transcript trans-spliced onto it. This way one ‘gene’ makes 2 mRNA (see diagram). This is not a conventional operon like those found in bacteria. This is not a minor thing in the nematodes genome – 15% of all genes in C. elegans are found in operons of this type. We still have monocistronic mRNA here so eukaryotes are behaving fairly well so far.

But in Drosophila and higher organisms dicistronic mRNAs have been found. No trans-splicing involved what so ever. This is definitely breaking one of the golden rules in biology. Here a promoter causes the transcription of 2 genes onto the same transcript. Each one of these gets translated. It is unclear at the moment if both genes have there own ribosome intonation site are if the ribosome that translates the first gene also translates the second one by not falling off at the stop codon of the first gene. One example (that is conserved between man and mouse) is the dicistronic mRNA coding the growth and differentiation factor 1 (GDF-1) and a membrane protein of unknown function (UOG-1). Whether they are co-transcribed because they have related function is obviously unknown.

Living creates are strange. We think we understand a bit about them, create rules that we think life follows so we can understand life better. But life doesn’t always follow the rules we think it does. So the best we can do is make rules or models up and test them as this is how science works. I just hate it when something I thought I new turned out to be wrong.

T. Blumenthal (2004). Operons in eukaryotes Briefings in Functional Genomics and Proteomics, 3 (3), 199-211 DOI: 10.1093/bfgp/3.3.199

Fanconi anaemia (FA) is an autosomal recessive chromosomal instability, characterised by congenital abnormalities, defective haemopoiesis, a high risk of leukaemia and development of solid tumours. FA patients have a 10% incidence of leukaemia and are 50 times more likely to develop solid tumours (particularly hepatic, oesophageal, oropharyngeal and vulval) by early adulthood. The disease consists of 13 complementation groups, each connected to mutations in 13 corresponding FANC genes (A, B, C, D1, D2, E, F, G, I, J, L, M, and N) which make up the FA pathway. Two thirds of FA cases are caused by inherited mutations in FANCA.

Interstrand crosslink damage

FA cells are characterised by hypersensitivity to damage by DNA Interstrand CrossLinking (ICL) agents such as mitomycin C and diepoxybutane. Cells fail to repair ICL damage, resulting in polysomies, radial chromosomal structures and unrepaired breaks (Figure 1). ICL agents tether two DNA strands together, creating a physical barrier to replication and transcription, which causes arrest of DNA replication. In normal cells, several mechanisms come in place to minimize damage and maintain the replication event. This necessitates removal of the physical barrier, repair of the gap and re-establishment of the replication fork. This complex repair mechanism involves the Fanconi anaemia proteins, translesion synthesis (TLS) polymerases, and the homologous recombination (HR) machinery. The FA pathway has also been implicated in spontaneous DNA damage repair, despite early speculation that the pathway was specific to ICL damage. In addition, there is some uncertainty as to whether the FA pathway also functions in intra-strand crosslink repair.

The FA pathway

The FA proteins are part of a complex DNA repair pathway, which is not yet fully understood. They are commonly arranged in three distinct groups according to their function. The first group is the FA core complex, which consists of FANC A, B, C, E, F, G, L and M. This complex stabilises stalled replication forks, processes the ICL site and activates a second group, which consists of FANCD2 and FANCI. Finally, group 3 consists of the remaining FANC proteins, BRCA2 (which is the same gene as FANCD1), FANCJ and FANCN, which have accessory or uncertain function in the pathway (Figure 2). The most recent model of this pathway is described in Wang (2007), Kennedy & D’Andrea (2005) and Niedernhofer (2007), and a detailed description of the model follows.

An ICL physically blocks replication by preventing separation of the strands by the advancing DNA helicase, which causes the enzyme to arrest. The replication fork arrest activates the ATR kinase, which subsequently phosphorylates FANCD2. A double strand break (DSB) upstream of the ICL is carried out by XPF-ERCC1, MUS81-EME1 or MUS81-EME2. Simultaneously, the monoubiquitin ligase FANCL, part of the FA core complex, monoubiquitinates FANCD2 and FANCI. This stage is regulated by the deubiquitinating enzyme USP1, and the monoubiquitin acts as a chromatin localisation signal. UPS1 is itself regulated by cell cycle controls, and self-cleaves in response to DNA damage. The integrity of the whole of the core complex is essential for this stage, as proven by the lack of chromatin localisation in FA core complex mutants. The downstream function of FANCI-Ub is uncertain, but FANCD2-Ub is arguably the most important component of this pathway, acting as a “fire captain” to recruit the proteins needed for the rest of the pathway (Figure 3).

As group 2 is activated, the FA core complex is also carrying out DNA processing in preparation for the next step in the pathway. FANCM acts as a DNA translocase. It anchors the rest of the FA core complex onto DNA, which stabilises the broken replication fork and facilitates removal of the ICL. A nucleotide excision repair (NER) endonuclease “unhooks” the ICL (which remains attached to the one strand), and translesion synthesis (TLS) polymerases recruited by the FA core complex fill in the gap. The TLS polymerases’ ability to pass through the damaged site has the trade-off of being error-prone, introducing random mutations in the repair site. The ICL adduct is then completely removed by a NER exonuclease, and the result is a replication fork broken by a DSB, with the ICL removed and the gap filled in (Figure 4). The next step is to repair the DSB, which is carried out by homologous recombination (HR) proteins.

HR-mediated replication fork re-establishment

FANCD2-Ub forms a complex with BRCA2 and chromatin. All the FANC proteins, along with BRCA1, NBS1, PCNA, RAD51 and other related proteins are recruited by the FANCD2-Ub/BRCA2-chromatin complex into nuclear foci at the damage site. BRCA1 forms a complex with BRCA2 and is essential for the formation of nuclear foci, even though its exact function is unknown. FANCJ, also identified as the helicase BRIP1 (BRCA1-interacting protein), depends on BRCA1 to translocate to the DNA damage site and unwind the replication fork in order to promote HR. The MRN complex, which consists of MRE11, RAD50 and NBS1 and has 3’-5’ exonuclease activity, processes either side of the DSB to prepare it for HR.

DSB repair can occur in two ways. Firstly by single-strand annealing, an error-prone non-homologous end joining (NHEJ) pathway, where the two strands are trimmed until homologous repetitive sequences are reached, and the strands are then annealed at that region of homology. This is mutagenic since the non-homologous intermediate region is deleted, but this is preferable to the arrest of replication. Secondly by strand invasion HR, where RAD51 is loaded onto chromatin by BRCA2 to form a nucleoprotein filament, within which Sister Chromatid Exchange (SCE) occurs and a Holliday junction forms between the strand being repaired and a homologous strand (Figure 5). Holliday junctions are intersections of four strands of DNA, which appear most commonly during meiosis when “crossing-over” occurs, and are resolved by endonucleases such as MUS81-EME1. FA cells show high levels of SCE in FANCC mutants, while other FANC mutant cells have normal SCE levels, suggesting a regulatory role for FANCC over SCE formation and a generally significant role in HR. HR thus re-establishes the replication fork and at the end of the S-phase, USP1 deubiquitinates FANCD2 to turn the pathway off.

In summary, ICL damage causes replication arrest, one of the strands is separated by a DSB, the FA core complex stabilises the broken fork and TLS repairs the ICL. At the same time, the structural integrity of the FA core complex is essential for the monoubiquitination of the “fire captain” FANCD2, which forms a complex with BRCA2 and chromatin and recruits HR and accessory proteins into nuclear foci. Finally, the MRN complex processes the ends of the DSB for HR to repair it and allow replication to restart.

There has been increased interest in the FA pathway in the past decade or so due to the discovery of its connection with cancer and complex relationship with the HR pathway. The pathway model is still in its early stages but already there are important questions raised about cell cycle regulation and its connection with DNA repair.

References

Kennedy, R.D. and D'Andrea, A.D. (2005) The Fanconi Anemia/BRCA pathway: new faces in the crowd. Genes Dev. 19: 2925-2940.

Niedernhofer,L.J., Lalai,A.S., and Hoeijmakers,J.H.J. (2005) Fanconi Anemia (Cross)linked to DNA Repair. Cell 123: 1191-1198

Patel,K.J. and Joenje,H. (2007) Fanconi anemia and DNA replication repair. DNA Repair 6: 885-890

Tischkowitz,M. and Dokal,I. (2004) Fanconi anaemia and leukaemia - clinical and molecular aspects. British Journal of Haematology 126: 176-191

Wang,W. (2007) Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins. Nat Rev Genet 8: 735-748